Entomo-pathogenic Fungi

Mass production of
Entomopathogenic fungi

Introduction
 Increasing public sensitivity to environmental pollution
and problems of pest resistance to chemical pesticides has
led to a global consensus to reduce or phase out extremely
noxious pesticides (e.g. methyl bromide).
 There is urgent need to make effort to develop new,
environmentally friendly crop protection strategies.
 At present, crop protection is trapped between the
increasing number of prohibited chemical pesticides and
the lack of safe, efficient alternatives. Entomogenous fungi
offer an environment friendly alternative to chemical
pesticides.

Entomopathogenic fungus (EPF)
 An entomopathogenic fungus is a fungus that
can act as a parasite of insects and kills or
seriously disables them.
 These fungi comprise a heterogenous group of
over 100 genera with approximately 750
species.
 Targets are distributed among 10 insect
orders:
 Hemiptera (59.6%), Coleoptera (40.9%),
Lepidoptera (17.5%), Thysanoptera (14.6%),
Orthoptera (9.4%), Diptera (7.0%),
Hymenoptera (2.9%), Isoptera (2.3%),
Siphonoptera (1.2%), and Blattodea(0.6%).

History
•In France and Italy, where silk production was important in the 16th and 17th
centuries, heavy losses of larval silkworms occurred from "muscardine disease. “
• In 1835, the Italian scientist Agostino Bassi de Lodi (the "Father of Insect
Pathology") showed that the muscardine disease of silkworms was actually
caused by a fungus that multiplied in and on the body of the insect.
B. Bassiana infected larva

Cond…
The muscardine fungus was the first microorganism to be
recognized as a contagious agent of animal disease which
was later identified as B. bassiana.
In 1883, Metchinikoff initiated mass culturing of fungus
and carried out the first experiment with two beetle pests.
 The green muscardine fungus, Metarhizium anisopliae was
described for the first time by Metschnikoff in 1879.
 Metarhizium anisopliae (Metschnikoff) Sorokin is the second
most widely exploited entomogenous fungus in biocontrol
after B. bassiana
 Verticillium lecanii (Zimm.) popularly called the “white holo”
is known to cause mycosis in a number of insects belonging
to the insect orders Homoptera, Coleoptera and
Lepidoptera.

Examples
Fly infected by
Entomophthora
fungus
 The asexual (anamorph) phases:
 Beauveria bassiana (against Colorado potato beetle,
cabbage looper, grasshoppers, silkworm,)
 Paecilomyces fumosoroseus (against white flies, thrips and
aphids)
 Metarhizium spp. (against soil insects like beetles,
locusts, white grub, spider mites)
 Verticillium lecanii (against white flies, thrips and aphids)
 Nomuraea
 Hirsutella (against mites)
 Entomophthora
The sexual (teleomorph) state:
 Cordyceps species that infect a wide
 spectrum of arthropods.

Life cycle
Dispersal of conidia from a larval
cadaver, attachment to the cuticle of a
new host, breaching the cuticle,
proliferation in the form of budding
hyphal bodies, germination and growth
of mycelium, formation of mycelial mass,
exit through the cuticle and formation of
new aerial conidia.

Mode of infection
 Attachment:- EGF have no known sexual cycle so insects are
infected by conidia (asexual propagules) which attach to the host
cuticle.
 Conidia germination:- in an environment with high humidity.
 Penetration and multipication:- The germ tubes developing from
the conidia penetrate the host cuticle and invade the haemocoel.
Mycelia proliferate and release blastospores by budding.
 Secretion of enzymes and toxin:- for degradation of proteins,
chitin and lipids in the insect integument.
 Death of insect:- Host insects are killed due to depletion of their
haemolymph nutrients and/or due to toxemia caused by fungal
toxic metabolites.
 Under moist conditions, the fungus emerges and produces a layer
of aerial conidia on the surface of host cadavers.

ENVIRONMENT INFLUENCE ON
PATHOGENICITY
 Of all the ecofactors that influence epizootic of a mycopathogen, the
most critical for sporulation, germination and invasion of the host is
high humidity (>90% RH).
 The rapidity of mycelial development and infection depends on
temperature.
 In general, optimum values fall between 20°C and 30°C (for example,
23°C for Beauveria brongiartii, 24°C for Entomophthora obscura, 25°C
for Beauveria bassiana and Nomuraea rileyi, 20°C - 25°C for Verticillium
lecanii and 27°C-28°C for Metarhizium anisopliae) .
 Conidia of M. anisopliae do not germinate at 93 % RH.

Microbial
control/Mycopesticides
 Mass multiplication of entomogenous fungi involves inundative
augmentation method, which is one of the three strategies of biological
control.
 It is application of the fungus, often in large amounts, for rapid short-
term control with no expectation of secondary infection .In this way,
the fungus is used in a similar way to a chemical insecticide.
 The terms “mycopesticide” or “mycoinsecticide” have been used to
describe this approach.

Advantages over chemicals
 They generally do not affect non target organisms so, extremely safe to
use.
 Eco-friendly and no residual toxicity.
 Relatively easy to mass produce (deuteromycetes fungi).
 Most commercial fungal products are formulated as spores, which are
easily adapted to existing application technology, like spraying.
 Have broad host range so control of multiple pests with the same
product is possible.
 Unlike other potential biocontrol agents, fungi do not have to be
ingested to infect their hosts but invade directly through the cuticle.

Mass production steps
Isolation & Culture Culture (nutrient media)
blastospore/conidia
Mass production harvested at 72 hrs
centrifugation (conc.)
Formulation filtered
dried paste
Storage aeration/ventilation
adjuvant added
stored
Production of adequate quantities of a good quality inoculum is an
essential component of the biocontrol programme.

Requirements/ considerations
 For successful and commercial productoin:
1. A fungal isolate must be selected with rapid growth, abundant
sporulation and high pathogenicity to the target pests.
2. Production costs must be minimal; medium used should be simple,
cheap, easily available, with easy production procedure.
3. Microbial products must be formulated to control different target
pests.
4. Formulated products must be suitable for long-term storage under
natural conditions without significantly losing viability and
infectivity.
5. Shelf-life is considered an important factor that determines the
commercial success of a biocontrol agent as well as its field efficacy.
6. An 18-month shelf-life is recommended for the agricultural market.

Isolation of pathogen
 B. bassiana and P. fumosoroseus are isolated from the diseased
caterpillar of S. litura collected from the fields.
 The diseased larvae (d.l) showed white colour for B. bassiana and
slight reddish mycelial surface growth for P. fumosoroseus.
 The (d.l) are surface sterilized with 0.1% mercuric chloride for few
seconds then thoroughly washed with sterilized double distilled
water.
 The excess water is removed by keeping the (d.l) on Whatman filter
paper No. 1.
 Diseased larvae were then cut into small pieces with the help of
sterile blade and the bits are aseptically transferred on to the
sabourand agar enriched with 1% yeast extract (SMYA) slants with
the help of sterile inoculation needle.
 The slants are kept at 25 ± 1oC.

Culture on media
Types:
Whole grain media
Whole grains viz Rice, Wheat, Raghi, Sorghum, Pearl millet
and Maize can be used for the sporulation of B. bassiana, P.
fumosoroseus and V. lecanii
Liquid media
Rice wash water, wheat wash water, coconut water and
rice cooked water are used for the growth and sporulation
of fungi.
Solid media
Non-synthetic solid media; carrot, ladies finger, jack seeds,
rice husk, and saw dust are used.

Suitable media
 Various agricultural products and by products such as grains,
vegetable wastes, seeds, rice husk, saw dust and liquid media such
as coconut water, rice and wheat washed water and rice cooked
water have been evaluated for mass production of 3 EPF:
 Beauveria bassiana, (Bals.) Vuil.
 Paecilomyces fumosoroseus (Wize) Brown and Smith
 Verticillium lecanii. (Zimm) Viegas.
 Among the grains, wheat supported maximum spore production for
B. bassiana while sorghum recorded maximum spore production in
P. fumosoroseus and V. lecanii.
 Similarly carrot, jack seeds and ladies finger also supported good
growth and sporulation of all the three tested fungi.
 Coconut water supported maximum growth and sporulation.

Production in Solid media
 100 g/ml of media is taken (washed well and soaked in
water overnight for grain, for rice 2-3hr)
 The excess water is drained by decanting and shade drying.
 Autoclave at 15 psi for 1 h; inoculation incubated at, 28-
300C for 15 days (under laminar air flow chamber)
 Shaking of flasks to avoid clumping, after 7 days of
inoculation.
 After incubation, 10 g/ml homogenized sample is taken for
sporulating bottle
 Transfer to 100 ml sterilized distilled water
 The flasks are shaken in mechanical shaker for 10 min
 The suspension is filtered through double layered muslin
cloth
 Counting of spores are made after the serial dilution of the
suspension using double ruled haemocytometer

Production in Liquid Media
 Three types of reactors or fermenters are commonly used:
1. The stirred tank
2. The tower and
3. The loop fermenters.
1.The stirred tank
 The stirred tank fermenter has the vertical cylinder with the agitator
mechanism placed centrally.
 These reactors produce a violent agitation of the culture medium with good
homogenization of the broth and a high gas transfer, thus avoid mycelial
aggregation and subsequent pellet formation.
 One drawback is damage to the mycelium on contact with the stirring
mechanism.
 Stirred tank fermenters have been employed for the production of the both
mycelium and yeast-like cells of all the common entomopathogenic fungi.

Cont…
2. The tower fermeter
 The tower fermenter is a vertical cylinder with a
height/diameter ratio greater than six
 Lack any mechanical agitation
 Nutrient mixing is promoted by the injection of
gas at the base of the reactor
 Most fungi produce mycelial aggregates in this
type of fermenter

Cont…
3. The loop fermenter
 The loop fermenter is a modification of the latter
in which the culture medium is forced back
down to the bottom of the reactor.
 The recycling of the medium is achieved by the
incorporation of a tube (internal recirculating or
by a pipe (external recirculating)
 Resting spores can be produced in this type of
fermenter.

Fermenters for the production of entomopathogenic
fungi
Diagram. A - Stirred tank fermenter, B - Tower tank fermenter, C and D loop
fermenter with internal (C) or external (D) recirculating of medium, E - Tray
reactor, F - homogeneous solid reactor, G - rotating disk fermenter: a - motor,
b - air sparger, c - heating and cooling system, d - impeller, e - baffle, f - foam
breaker, g - rotating discs, h - granular media.

Types of fermentations
1. Solid substrate fermentation (SSF)
 Uses solid substrate for mass multiplication.
Advantages:
1. Yields conidia (natural sporulation by mycelia)
2. Conidia obtained is stable (not heat sensitive)
3. More infectious than blastospores.
Disadvantages:
1. Yield is low
2. Problem in harvesting

conti…
2. Submerged fermentation
 It is designed to take advantage of existing industrial
fermenters which gives higher yield in liquid media.
 Using a mineral medium (0.9% NaNO3, 0.25% KH2PO4,
0.075% MgSO4, 1.25% CaCl2) supplemented with 1%
sucrose, a yield of 3-5 X 108 conidia/ ml is obtained after
72-h fermentation in fermenters up to 63 m3 capacity, at
26-28°C, with proper aeration
 Inclusion of peptone in the medium result in an average
yield of 2-3 X 109 spores ml/L.

Advantages:
1. Yield is high
2. Harvesting is easy
3. Cheaper
Disadvantages:
 Often gives rise to blastospores or a mixture of
blastospores and conidia.
 Blastospores show shorter viability (suceptibility to
drying)
 Less infectious than conidia

conti…
3. Diphasic fermentation
It combines the advantages of both solid and liquid
media
 The fungus is allowed to grow in fermenters up to the
end of the log phase for maximal production of
mycelial biomass
 Subsequently transferred on to inert substrates in
trays for the production of aerial conidia in the form
of natural inoculum

Advantage:
 It is simple
 it is to increse growth of conidia harvest
Disadvantages:
 Considered to be the most expensive and
labour-intensive
 Unsuitable for conventional processing of
fungal material in fermenters

Green muscardine fungus
 The colony of M. anisopliae
appears white when young but
as the conidia mature, the colour
turns to dark green.
 A chain of conidia is formed on
each conidiophore
 Effective in the suppression of
soil borne pests.

Light micrograph of
submerged spores of M.
anisopliae var. acridum

 Production procedure
The fungus can be mass produced in
conventional laboratory media as well as on
crushed grains. The cheapest media till date
known are:
 (1) Cassava chips mixed with rice bran
supplemented with urea or fish meal extract
 (2) Coconut water wasted from copra making
industry
 (3) carrot broth.

Host insets of Metarhizium anisopliae
Helicoverpa armigera
Dimond Black Moth
Brown plant hopperRoot grub
Locusta migratoria infected
with Metarhizium anisopliae

The mortality of Helicoverpa armigera applied
with Metarhizium anisopliae
1. Healthy larvae (8 days old larvae)
2. Dead larvae ( 5 days after spore application)
3. Dead larvae with thick fungal mat (7 days after spore application)
4. Dead larvae covered with green coloured conidia (10 days
after spore application)

Mass production
1. On coconut water (liquid media)
 Coconut water (40 ml) contained in 375 ml liquor bottles
plugged with cotton plug are sterilized in batches of 9-10
bottles in 12 litre pressure cooker for 20 minutes at 15
psi.
 The bottles are inoculated with 1 ml suspension
containing spores of the fungus with the help of a sterile
injection syringe.
 The bottles are inoculated in a laminar flow chamber and
rested on flat surface for 2 days or till the surface of
medium is fully covered by the olive green sporulated
fungus.
 The whole culture is crushed thoroughly in an ordinary
mixer and used in the field.
 From a single average size coconut , 5-6 bottles of cultures
can be made.

2)On rice(solid media)
Culture on
rice
 Rice grains continue to be the most used substrate for
conidial production.
 Most companies use from 300 to 500 g of rice per plastic
bag.
 Rice is soaked in water, drained and transferred to plastic
bags following sterilization
 Conidial suspensions (5 ml suspension/300g rice bag) are
used to inoculate.
 Bags are hand mixed to distribute the inoculum evenly and
to disaggregate clusters of rice for adequate colonization
and good sporulation.
 Incubation temperatures range from 25 - 300C for 14
days.
 During the dry season, this procedure takes 56 days for
satisfactory dehydration and conidia can then be
harvested.

3) Production by diphase system
In Brazil and Russia-
 This involves submerged fermentation using corn extract and
molasses with continuous aeration
 After 40-50hr resulting mycellial mass and biomass is transferred into
stored panes or trays. Then incubated for 6 days.
 Yield- 5 to 7.5 x 1012/ kg of dried preparation.
In Australia –
 Granular bit formulation by 2 phase method.
 10,000 to 12000 L of liquid medium(Agricultural waste, sucrose, Ca,
water) are inoculated with blastospore
 Incubation for 5 days in a fermenter
 Yield 4x 1012 conidia/kg bait

Ideal condition for mass production
 The spore count, radial growth, sporulation and biomass were
maximum when M. anisopliae were cultured both Czapeck’s dox agar
and potato dextrose agar media
 Highest sporulation of M. anisopliae observed when cultured in wheat
(8,300x104). Hence, it is recommended as best solid media for the
mass production of M. anisopliae
 The optimum temperature-25-30°C and
Ideal pH 7.0 for the mass production
 High-density polyethylene (HDPE) bags are ideal and also cost
effective and give good result for the mass production of M. anisopliae

B. bassiana
The fungus is called as
white muscardine
fungus.
 The fungus spores and
mycelia are milky white
and found sprouting on
the body of lepidopterous
insects like Helicoverpa
armigera, Spodoptera
litura.
White muscardine fungus Conidia on conidiophores

Conti…
 Helicoverpa armigera larvae
infected with Beauveria bassia
B. bassiana on S. litura larval cadavers

Culture and Mass production
 Culture
 B. bassiana is easily cultured on solid and liquid media, e.g.
Saubouraud or potato dextrose agar and broth.
 Streptomycin (500 mg/l) must be added to agar to obtain a
pure culture free from any bacteria.
 Seed inoculum is better prepared as a submerged culture in
seed fermenter or in Erlenmeyer flasks on shaker.
 Aerial conidia are produced on solid media.
Mass production
 At present, mass production of B. bassiana is generally based
on diphasic and submerged fermentation techniques.

Mass production
On carrot
 Carrot cut into small pieces (40 g) is
washed in potable water and transferred
to conical flask (250 ml) and 15 ml of
distilled water is added.
 The conical flasks are plugged with
cotton and autoclaved for 20 min at 15
psi.
 The flasks are allowed to cool and taken
to laminar flow chamber for inoculation.
 From a clean uncontaminated mother
culture in slant loopful quantities of B.
bassiana spores are transferred
aseptically.
 The flasks are incubated at room
temperature. The spores are obtained in
a fortnight
 Beauveria bassiana cultured on solid substrates in polypropylene
bags
Beauveria bassiana cultured on solid
substrates in polypropylene bags

On cereal
 B. bassiana is cultivated on different media:
 wheat bran, wheat hybrid, oat semolina.
 The mass of 30g of media with 45 ml water is poured
to glass bowls.
 The treatment sterilized for 30 minutes at 120oC and
pressure 1 mPa/cm2.
 The media, after cooling to approx. 35oC, is inoculated
by the spores from the agar (PDA) or by blastospores
and mycelium from a seed fermenter.
 The culture is stored in a thermostat at 25oC +/- 1oC
and 75 % R.H.

White halo fungus, Verticillium lecanii
 V. lecani commonly called as
white halo fungus.
 Dead larvae generally mummify
due to fungal infection. The
cadavers show an white mycelial
growth on the insect surface
(except on the head capsule)
 The fungus is known to cause
epizootic when the
environmental conditions are
favourable. “
Aphid infection
Spirilling whitefly infection

Production procedure
 The fungus is multiplied on cheap media for large scale
production.
 On Sorghum: grains devoid of pesticide residues (40 g) is washed
and transferred to conical flask (250 ml) and 15 ml of distilled
water is added.
 The conical flasks are plugged with cotton and autoclaved for 20
min at 15 psi.
 The flasks are allowed to cool and taken to laminar flow chamber
for inoculation.
 From a clean uncontaminated mother culture in slant loopful
quantities of V. lecani spores are transferred aseptically.
 The flasks are incubated at room temperature. The spores are
obtained in a fortnight.

Contd….
 Paecilomyces farinosus
 The diseased larvae show slight
reddish mycelial surface.
 It is mass produced on wheat bran,
sporulated fungus is harvested from
substrate, dried and applied in soil
granular form.
Nomuraea rileyi
 For control of cabbage and soybean
looper, it is grow on solid
media(Agar).
 Yield- 6.3x108 conidia/cm2 of surface.

Formulation and stability
 Mycopesticides are defined as products based on living fungal
propagules intended to control pests through inundative or
inoculative applications.
 To enhance stability and improve the handling of the conidia,
2 types of formulations are prepared:-
1) The product as a powder in heat-sealed plastic-lined foil sachets,
the addition of skimmed milk powder improves longevity by
absorbing humidity, and functions as a sticker and sunscreen after
application.
2) Product is prepared with mineral oil that contains 1.2% emulsifier.
The conidia are mixed with Ultra-Fine Spray Oil @ 1.25×108
conida/ml oil.
 This type of formulation has shown better stability both at room
temperature and under refrigeration, which is important for retail
sale.

Formulation types
 Wettable powder (WP):- ‘‘A powder formulation to be
applied as a suspension after dispersion in water.’’
 Granule (GR). ‘‘A free-flowing solid formulation with
particles of controlled and uniform size range ready for
use and with the active ingredient strongly adhered to or
incorporated into the granule.
 Water dispersible granule (WG). ‘‘A formulation
consisting of granules to be applied after disintegration
and dispersion in water.’’
 Contact powder (CP). ‘‘Insecticidal formulation in powder
form for direct application.’’
 Free-flowing powders suitable for dusting are termed
dustable powders (DP).

Cond…
 Suspension concentrate (SC). ‘‘A stable suspension of active
ingredient(s) in water, intended for dilution with water before use.’’
 oil miscible suspension. ‘‘A stable suspension of active
ingredient(s) in a fluid intended for dilution in an organic liquid
before use.’’
 Ultra-low volume (ULV) suspension (SU). ‘‘A suspension ready
for use through ULV equipment.’
’
 Oil dispersion (OD). ‘‘A stable suspension of active ingredient in a
water-immiscible fluid, which may contain other dissolved active
ingredient(s), intended for dilution in water before use.’’

Common formulation types
 Technical concentrates in the form of fungus-colonized
substrates (26.3%), wettable powders (20.5%), and oil
dispersions (15.2%).
 The remaining types include granules (2.9%), technical
materials (2.9%), baits (1.8%), water dispersible granules
(1.8%), oil miscible flowable concentrates (1.2%), ULV
suspensions (0.6%), suspension concentrates (0.6%),and
contact powders (0.6%).
 Of the over 750 species of fungi known to be pathogenic to
insects, six have been commercialized and the cosmopolitan
pathogens, such as B. bassiana, M. anisopliae are the best
known so far.

Doses and application
 As an insecticide, the spores are sprayed on affected
crops as an emulsified suspension or wettable powder
 The Metarhizium-based mycoinsecticide conidia are
applied by aerial spraying which should be mixed with
diesel oil or kerosene before spraying at a rate of 2.5–
5×1012 conidia/ha to control the spittlebugs.
 Boverin if applied alone 3 to 20 kg/ha(uneconomic). So 2
kg boverin+ ¼ insecticides(400g)/ha.
 Soil application on B. brongniartii blastospore-
2x1013/ha
 Aerial spray of metaquino (M.anisoplae)@ 6x1011 to
1.2x1012 conidia/ha causes 65% mortality

Commercial Products
Of B. bassiana
 Since the late 1980s, focus has been on the development of B.
bassiana as a commercial mycoinsecticide by Mycotech.
 In 2000, Mycotech was taken over by Emerald Bio Agriculture,
who continue to market these fungal-based formulations.
 Boverin, was developed in the USSR during the 1970s . It has
been used extensively to control the Colorado potato beetle, the
larvae of codling moth
 “Mycotrol” was fully registered in 1999 by the EPA of the USA. Its
WP formulation cause high mortality of aphids, whiteflies, thrips
on tomato and ornamental crops.
 “Metaquino” has been in use in Brazil. It was also used against
coffee berry borer in Brazil and coconut leaf beetle in Taiwan.

Others
 Metarhizium anisopliae is registered in the U.S. for control of
cockroaches as “Green Muscle”.
 Verticillium lecanii sold as ‘’Vartalec’’. It is produced by liquid
fermentation as blastospores which are formulated with a
nutrient source in a wettable powder
 Hirsutella sold as Mycar.
 Recent advances by Koppert in formulation technology have
resulted in an adjuvant based on an emulsifiable vegetable oil,
called “Addit”, which can enhance activity of “Mycotrol” at low
humidities
 Paecilomyces fumosoroseus Strain 97 has been approved for use
on ornamentalsto manage whiteflies, aphids, thrips, and spider
mites and is sold by Thermo Trilogy Corp.

Commercial products in India
 B. bassiana – Bio power, Biogard
 Metarhizium anisopliae- Biomagic, Biomet
 Verticillium lecanii- Biovert, Biocatch
 Hirsutella- Mete hit, Mycohit
 Paecilomyces fumosoroseus- Paci Hit

Limitations and disadvantages
 The major issues involved in mass production and utilization of
mycopathogens are selection of effective strains, development of cost
effective methods for mass rearing, development of effective methods for
storage and shipment and creation of effective formulation.
 High concentrations of spores are often needed to get adequate control.
 The kill time is relatively long (~1 week for most fungi).
 Their broad host range can sometimes be a problem, for beneficial
insects.
 Environmental factors like temperature, humidity and sunlight play a
profound role on the field persistence of entomopathogenic fungi.
 Prolonged exposure to sunlight can also inactivate spores, reducing
persistence in the crop.

Major technical hurdles
 They include:
 Improvements in screening and quality control to ensure
that only viable, virulent inoculum reaches the market
 Development of media to increase the virulence and
ecological fitness of conidia
 Improved formulations to increase both the virulence and
shelf life of the inoculum
 Better application strategies to ensure that sufficient
inoculum contacts the pest because mortality is dose-
related above certain thresholds

Future prospects
 Currently, a yield of submerged conidia in much lower
quantities than blastospores remains an obstacle to the use
of the submerged culture method for commercial production
of conidia
 Attempts to use these fungi as bioinsecticides have usually
failed as they are too difficult to mass produce and are often
ineffective under conditions of moderate humidity in the
field
 Thus, genetic engineering may help in improving efficacy of
mycoinsecticides but, all hazards must be assessed, step by
step concerning all potential dangers to non-target
organisms before release into the environment.

Conclusion
 For mass production fungal isolate must be selected with
rapid growth, abundant sporulation and sufficiently high
pathogenicity to the target pests.
 production costs must be minimal; medium should be
simple in composition, cheap in price and available in large
quantities with easy production.
 Formulated products must be suitable for long-term
storage under natural or nearly natural conditions without
significantly losing their viability and infectivity.
 Shelf-life is considered a pivotal factor that determines the
commercial success of a biocontrol agent as well as its field
efficacy.

References
 Soundarapandian, P. and R. Chandra, 2007. Mass production of
endomopathogenic fungus Metarhizium anisopliae
(Deuteromycota; Hyphomycetes) in the laboratory. Res. J.
Microbiol., 2: 690-695.
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