CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2011 6, No.

056

Review

Microbial biopesticides: opportunities and challenges

Opender Koul*

Address: Insect Biopesticide Research Centre, 30- Parkash Nagar, Jalandhar 144003, India.

*Correspondence: Email: okoul©airtelmail.in; okoul©koulresearch.org

Received: 10 October 2011

Accepted: 17 November 2011

doi: 10.1079/PAVSNNR20116056

The electronic version of this article is the definitive one. It is located here: http://www.cabi.org/cabreviews

© CAB International 2011 (Online ISSN 1749-8848)

Abstract

Pesticides based on microorganisms and their products have proven to be highly effective, species
specific and eco-friendly in nature, leading to their adoption in pest management strategies around
the world. The microbial biopesticide market constitutes about 90% of total biopesticides and
there is ample scope for further development in agriculture and public health, although there are
challenges as well. This article reviews the various microbial biopesticides that are commercially
available, the different approaches for their production and development, the recent technological
advances and the challenges faced by the microbial biopesticide field in the future.

Keywords: Microbials, Bacteria, Viruses, Fungi, Nematodes, Biopesticides, Pest management,

Commercialization

Introduction and certain minerals) is increasingly being adopted. North

The damage and destruction inflicted on crops by pests
have had a serious impact on farming and agricultural
practices for a long time. These pests include insects,
fungi, weeds, viruses, nematodes, animals and birds. It has
been estimated that nearly 10 000 species of insects,
50 000 species of fungi, 1800 species of weeds and 15 000
species of nematodes destroy food and fibre crops used
by millions of people worldwide. In India alone, 30% of the
crop yield potential is lost as a result of insects, disease
and weeds, corresponding to 30 million tons of food grain.
In an attempt to avoid such losses, the primary strategy
employed has been to eliminate the pests by using
chemical pesticides such as chlorinated hydrocarbons,
organophosphates and carbamates. However, despite
the successes achieved, potential hazards or risks have
emerged that have had a substantial impact on the
environment; compounded further by indiscriminate and
excessive use of the products. Consequently, beneficial
species have been lost and residual problems have
increased, with subsequent impact on the food chain,
groundwater contamination and resistance in pests. To
overcome the hazards associated with chemical pesti-
cides, the use of biopesticides (pesticides derived from
such natural materials as animals, plants, microorganisms
America uses the largest percentage of the biopesticide
market share at 44%, followed by the EU and Oceania
with 20% each, South and Latin American countries with
10% and about 6% in India and other Asian countries
[1, 2]. In terms of sales of biopesticides, the global market
in 2007 was US$672 million and projected as US$1000
million for 2010 (Figure 1). The current growth of the
chemical pesticide market is about 1-2% per year, while
growth in microbial pest control is about 10% per year
(with some estimates projecting growth as high as 20%)
[3]. As of 2007/08, estimates of microbial biopesticide
sales were US$396 million at the end-user level, although
these estimates are likely to be just a fraction of the total
usage of such products, owing to the lack of information
available on the non-commercial use of such products in
these regions. Such estimates could be more unreliable
globally, given unquantified sales in other parts of the
world. A recent survey has shown that Europe is a large,
intense and diverse pesticide market valued at $12850
million in 2008; approximately 31.7% of the world's total.
The six largest national agrochemical markets are in
France, Italy, Spain, UK, Germany and Turkey. France
alone accounts for 26.2% of agrochemical sales of the
wider Europe, with Italy taking 19.8%. The Nordic coun-
tries, on the other hand, consume comparatively small

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2 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
2007 2010
1000
400
See
700
fl S In view of various opportunities and challenges that
0 600
vi..E
400
300
-ea
Zan ico 12D
100
0
Glob Eurup, Asia
Figure 1 Biopesticide market in different continents
(Source: BCC Research Report 2010)
amounts; Denmark, Estonia, Finland, Lithuania, Norway
and Sweden combined use just 2.2% of the total [4].
Over the past 150 years, a great deal of knowledge has
been gathered on the use of microorganisms including
bacterial, fungal, viral, protozoan or nematode-based
preparations as pest control agents (see Box 1). However,
the widespread use of biopesticides has been restricted,
owing to various constraints at the developmental,
registration and production levels. Assessment of the use
of microorganisms in pest management suggests that
advances have been incremental, rather than transfor-
mative. This is apparently the result of higher production
costs in comparison with conventional chemical pesti-
cides, narrow target-species ranges and inefficient delivery
systems. Although there have been ample advances in
terms of new discoveries of microbial isolates and an
increasing ability to genetically manipulate the microbial
agents involved, concerns about pest resistance and
environmental and human safety remain. Furthermore,
increased adoption of microbial biopesticides has come
under threat from the development of new biorational
pesticides (including pest control agents, and chemical
analogues of naturally occurring biochemicals such as
pheromones, insect growth regulators, etc., which are
more environment friendly than synthetic chemical
pesticides). Comparative analyses of conventional insec-
ticides with microbial biopesticides (see Box 2) suggest
that there is still a lack of knowledge regarding the
interactions of microbial biopesticides with pests, natural
enemies and the wider ecosystem.
Microbial control agents, based on naturally occurring
fungi, bacteria, viruses or nematodes have offered some
realistic alternatives to chemical pesticides when used
as part of an ecologically based integrated pest manage-
ment (EBIPM) or area-wide pest management strategy
(AWPM) [5, 6]. There are many reasons for the recent
increased interest in microbial biopesticides, including
the development of resistance to conventional synthetic
pesticides, a decline in the rate of discovery of novel
insecticides, increased public perception of the dangers
associated with synthetic pesticides, host-specificity of
microbial pesticides and improvement in the production
and formulation technology of microbial biopesticides.
are associated with the development of microbial bio-
pesticides, this article reviews the commercially available
microbial biopesticides, the different approaches for their
production and development, the technological advances
made and constraints envisaged in future in the field of
microbial biopesticides.
LatinAmerica
Microbial Biopesticides in Pest Management
Out of all the biopesticides used today, microbial bio-
pesticides constitute the largest group of broad-spectrum
biopesticides, which are pest specific (i.e., do not target
non-pest species and are environmentally benign). There
are at least 1500 naturally occurring insect-specific
microorganisms, 100 of which are insecticidal [2]. Over
200 microbial biopesticides are available in 30 countries
affiliated to the Organization for Economic Co-operation
and Development (OECD) [7]. There are 53 microbial
biopesticides registered in the USA, 22 in Canada and
21 in the European Union (EU) [8, 9] although reports of
the products registered for use in Asia are variable [10].
Overall, microbial biopesticide registrations are increasing
globally, the expansion of various technologies has
increased the scope for more products and the change in
the trend to develop microbial products is definitely on
the rise [1, 11].
Bacterial Biopesticides
The bacteria that are used as biopesticides can be divided
into four categories: crystalliferous spore formers (such
as Bacillus thuringiensis); obligate pathogens (such as Bacillus
popilliae); potential pathogens (such as Serratia marcesens);
and facultative pathogens (such as Pseudomonas aeruginosa).
Out of these four, the spore formers have been most
widely adopted for commercial use because of their safety
and effectiveness. The most commonly used bacteria are
B. thuringiensis and Bacillus sphaericus. B. thuringiensis is a
specific, safe and effective tool for insect control [12]. It
is a Gram-positive, spore-forming, facultative bacterium,
with nearly 100 subspecies and varieties divided into 70
serotypes [13]. The insecticidal property of B. thuringiensis
resides in the Cry family of crystalline proteins that are
produced in the parasporal crystals and are encoded by
the cry genes. The Cry proteins are globular molecules
(65-145 kDa, depending on the strain) with three struc-
tural domains connected by single linkers. The 200 Cry
proteins belong to a single family that contain about
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Box 1 Historical Perspective
Diseases of honeybees and silkworm have been known in
China and India since ancient times. Observations of diseased
silkworms were recorded in China as far back as 2700 B.C.
The first documentation of insect diseases is usually attrib-
uted to the descriptions of honeybee maladies recorded by
Aristotle somewhere between 330 and 323 B.C. Most of
the earlier work on insect pathology is devoted to these
two domesticated insects, viz. the silkworm, Bombyx mori
(Linnaeus) and the honeybee, Apis mellifera Linnaeus. In 1835,
Agostino Maria Bassi (called the Father of Insect Pathology)
published his work on white muscardine disease of the silk-
worm and the fungus responsible for this disease was later on
named as B. bassiana (Balsamo) Vuillemin [135].
The first published record of a diseased insect appears to
be that of the 'Chinese plant worm', described and illustrated
by Rene-Antoine Ferchault de Reaumur in 1726. He under-
stood the `stemlike vegetable growth', emerging from a
noctuid larva to be the root of a plant. In 1749, a Franciscan
friar in Cuba described dead wasps with little trees growing
out of their bellies. This was a fungus now known as a
member of the genus Cordyceps [136]. During the mid-
nineteenth to early-twentieth centuries, numerous scientists
reported on the biology and pathology of the entomo-
pathogenic fungi who recommended study of fungal epizootics
of insect to determine the most effective means of introdu-
cing and transmitting such pathogens.
A Russian entomologist, Metschnikoff, conducted the
first systematic experiments on the control of injurious
insects with microorganisms by infecting grubs of the grain
beetle, Anisoplia austriaca, with the green muscardine fungus,
M. anisopliae (Metschnikoff) Sorokin, in 1879. The fungus was
found to be even more effective against the sugar beet
curculio, Cleonus punctiventris (Germ.). A Japanese scientist,
who named it Bacillus sotto, first discovered a bacterium in
silkworms in 1901 but it was 10 years later that a German
50 subgroups [14]. This three-domain family is charac-
terized by protoxins of two different lengths, one being
longer with a C-terminal extension necessary for toxicity.
This extension also has a characteristic role in crystal
formation within the bacterium [15]. Cry proteins are
responsible for feeding cessation and death of the insect
and their biology has been comprehensively described [2].
Cry protoxins are ingested [13, 16] and then solubilized
(some toxin families need more alkaline pH and others
respond more to neutral pH), releasing a protease-
resistant biologically active endotoxin, before being
digested by protease of the gut to remove amino acids
from its C- and N-terminal ends. The C-terminal domain
of the active toxin binds to specific receptors on the
brush border membranes of the midgut followed by the
insertion of the hydrophobic region of the toxin into
the cell membrane [17]. This creates a disruption in the
osmotic balance, because of the formation of trans-
membrane pores and ultimately cell lysis occurs in the gut
wall, leading to leakage of the gut contents (Figure 2).
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Opender Koul 3
scientist, Ernst Berliner, isolated the bacterium from diseased
flour moths and assumed it was responsible for the sudden-
collapse disease, which was affecting the insect. Since he had
obtained the first sample from a mill in Thuringia, he called
the bacterium Bacillus thuringiensis (Bt).
Field trials with Bt to control the European corn borer
were conducted as early as the late 1920s and in 1938 the
first commercial Bt preparation (Sporeine) came on to the
market in France. However, it was not until the 1960s that its
use became widespread. In 1964, Biospor became the first Bt
preparation to be licensed as a pesticide in Germany. Another
breakthrough in the development of microbial control came
with the discovery and practical application of the milky
disease bacteria, Bacillus popilliae Dutky, for the control of the
Japanese beetle, Popillia japonica Newman on turfs in USA
during 1940s [137]. But with the discovery in 1970 of the
particularly virulent Bt strain B. thuringiensis kurstaki, which is
effective against the larvae of certain butterflies and moths,
and the discovery in 1983 of the B. thuringiensis tenebrionis
strain, which is effective against certain beetles, including the
Colorado potato beetle, the range of microbial agents avail-
able has grown considerably.
Viruses as microbial pesticides have a history of slow
development. A. Maestri and E. Cornalia made the first
observations of viral infections in 1856. Hoffman reported
NPV of nun moth in 1891 and Bolle demonstrated viral
transmission from diseased to healthy insects in 1894. The
first description of OBs as causatives of infection came
in 1906 and in 1918 the filterable and transmissible nature
of viral infection was established. However, the first bio-
chemical study of the OBs was published in 1934 by
Glaser and Stanley and it was after 1947, Edward Steinhaus
in USA and Gernot Bergold in Canada undertook a
major effort to develop insect viruses as microbial insecticides
[138].
This induces starvation and lethal septicaemia of the
target pest.
Many details of this process are still not understood
and the action seems to be more complex as indicated by
the existence of novel receptors and signal transduction
pathways induced within the host following intoxication
[18]. This could lead to altered activation of midgut
proteases, resulting in differences in the toxin structure
that could affect binding to the peritrophic membrane,
thereby accounting for host specificity [19]. Subspecies
of B. thuringiensis that are used as biopesticides include
B. thuringiensis tenebrionis (targeting Colorado potato
beetle and elm leaf beetle larvae), B. thuringiensis kurstaki
(targeting a variety of caterpillars), B. thuringiensis israe-
lensis (targeting mosquito, black fly and fungus gnat larvae)
and B. thuringiensis aizawai (targeting wax moth larvae
and various caterpillars, especially the diamondback moth
caterpillar). These bacteria are mass-produced through
either solid or liquid fermentation. The cost of one
litre of medium for the production of B. thuringiensis
4 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
Box 2 Advantages and Disadvantages of Microbial Versus Chemical Pesticides
Microbial Pesticides
Advantages
Microbial pesticides are non-toxic and non-pathogenic to
non-target organisms and the safety offered is their
greatest strength.
Action of microbials is specific to a single group or species
of pests, therefore, do not affect directly beneficial animals
such as predators and parasitoids.
Microbial pesticides can be used in many habitats where
chemical pesticides have been prohibited. Such habitats
include recreational and urban areas, lake and stream
borders of watersheds, and near homes and schools in
agricultural settings.
Residues of microbial pesticides are non-hazardous and are
safe all the time, even close to harvesting periods of the
crops.
They have a potential to control vectors. Some pathogenic
microbes can establish in a pest population or its habitat
and provide control during subsequent seasons or pest
generations.
Disadvantages
Owing to the specificity of the action, microbes
may control only a portion of the pests present in a field
and may not control other type of pests present in treated
areas, which can cause continuous damage.
As heat, UV light and desiccation reduces the efficacy of
microbial pesticides, the delivery systems become an
important factor.
Special formulations and storage procedures are
necessary. Shelf life is a constraint, given their short shelf
lives.
Given their pest specificity, markets are limited.
The development, registration and production costs
cannot be spread over a wide range of pest control
sales; for example, insect viruses are not widely avail-
able.
israelensis has been estimated as US$ 1.2 and 0.01 using
commercial complex medium versus by-products of in-
dustrial factories, respectively. Some common commer-
cial products based on Bacillus thuringiensis are available
globally (Table 1).
B. sphaericus is a Gram-positive strict aerobic bacter-
ium, which produces round spores in a swollen club-like
terminal or subterminal sporangium [20]. B. sphaericus
strains were isolated in the mid-1960s from mosquitoes,
blackflies and grasshoppers [21]. These bacteria produce
an intracellular protein toxin (5511-1) and a parasporal
crystalline toxin at the time of sporulation [22]. The
mosquito-larvicidal binary toxin produced by B. sphaericus
is composed of BinB and BinA, 51.4 and 41.9 kDa,
respectively. Bin proteins in combination form a crystal
and in solution can exist as an oligomer containing two
copies each of BinB and BinA [23, 24]. Some toxic strains
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Some insects develop resistance to several insect patho-
gens. Resistance management will have to be practiced, as
it is with chemical pesticides.
Chemical Pesticides
Advantages
Chemical pesticides are cost-effective and economical to
control pests. Low labour input is required and they allow
large areas to be treated quickly and effectively. Estimates
suggest that 4-fold returns are expected after the use of
these pesticides.
Chemical pesticides are flexible in the sense that they
control all pests with variation in type, activity and per-
sistence.
They are easily available in large quantities, at high quality
and at reasonable price.
Pesticides are often used to stop the spread of pests in
imports and exports, preventing weeds and protecting
households from destruction.
They have substantial application in protection of pets and
humans from pests.
Disadvantages
Reduction in beneficial insects due to the toxicity of these
pesticides to non-target pests can result in changes in
biodiversity of an area and affect natural biological balance.
Drift of sprays and vapour of chemical pesticides can cause
severe problems in different crops, waterways and general
environment.
Chemical pesticides do leave residues in food, either by
direct application or by bio-magnification. Because of their
persistent use in agriculture, chemicals can reach under-
ground aquifers and contaminate water bodies.
There are poisoning hazards for pesticide operators given
excessive exposure; though it depends on dose, toxicity,
sensitivity and duration of exposure.
Overuse of chemical pesticides encourages resistance.
also produce 100 kDa toxins encoded by mtx genes [25].
Bacillus-sphaericus-based products are commonly used for
mosquito control (Table 1).
Other species of bacteria have little impact on pest
management though some commercial products based on
Agrobacterium radiobacter, B. popilliae, B. subtilis, P. seudo-
monas cepacia, P. seudomonas chlororaphis, P. seudomonas
flourescens, P. seudomonas solanacearum and P. seudomonas
syringae are available (Table 1).
Viral Biopesticides
Over 700 insect-infecting viruses have been isolated,
mostly from Lepidoptera (560) followed by Hymenoptera
(100), Coleoptera, Diptera and Orthoptera (40) [2].
About a dozen of these viruses have been commercialized

for use as biopesticides (Table 1). The viruses used for
insect control are the DNA-containing baculoviruses
(BVs), Nucleopolyhedrosis viruses (NPVs), granuloviruses
(GVs), acoviruses, iridoviruses, parvoviruses, polydna-
viruses, and poxviruses and the RNA-containing reo-
viruses, cytoplasmic polyhedrosis viruses, nodaviruses,
picrona-like viruses and tetraviruses. However, the main
categories used in pest management have been NPVs and
GVs. These viruses are widely used for control of vege-
table and field crop pests globally, and are effective against
plant-chewing insects. Their use has had a substantial
impact in forest habitats against gypsy moths, pine
sawflies, Douglas fir tussock moths and pine caterpillars.
Codling moth is controlled by Cydia pomonella GVs on
fruit trees [26] and potato tuberworm by Phthorimaea
operculella GVs in stored tubers [27]. Virus-based prod-
ucts are also available for cabbage moths, corn earworms,
cotton leafworms and bollworms, beet armyworms,
celery loopers and tobacco budworms (Table 1).
The mechanism of viral pathogenesis is through repli-
cation of the virus in the nuclei or in the cytoplasm
of target cells. The expression of viral proteins occurs
in three phases. First is the early phase, i.e. 0-6 h post-
infection, second is the late phase, i.e. 6-24 h post-
infection and the third phase is very late phase, i.e. up to
72 h post-infection. It is at the late phase that virions
assemble as the 29 kDa occlusion body protein is syn-
thesized. Numerous virions of NPVs are occluded within
each occlusion body to develop polyhedra. However,
the GV virion is occluded in a single small occlusion body,
to generate granules (Figure 2). Infected nuclei can pro-
duce hundreds of polyhedra and thousands of granules
per cell. These can create enzootics, deplete the pest
populations, and ultimately create a significant impact on
the economic threshold of the pest.
The viral biopesticides are usually only active against
a narrow host spectrum and after their application to
plant surfaces, and baculovirus occlusion bodies (OBs)
are rapidly inactivated by solar ultraviolet (UV) radiation,
particularly in the UV-B range of 280-320 nm [28].
However, their efficacy can be improved by the use of
formulations that include stilbene-derived optical bright-
eners, which increase susceptibility to NPV infection by
disrupting the peritrophic membrane [29] or inhibiting
sloughing [30] or virus-induced apoptosis of insect midgut
cells [31]. UV inactivation could be controlled by creating
systems, which can filter UV radiation, as has been
demonstrated by using plastic greenhouse structures that
reduced the intensity of incident UV-B (280-315 nm)
readings by >90% compared with external readings leading
to an increase in the prevalence of infection in larvae [32].
Fungal Biopesticides
The pathogenic fungi are another group of microbial pest
management organisms [2] that grow in both aquatic as
well as terrestrial habitats and when specifically associated
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Opender Koul 5
with insects are known as entomopathogenic fungi. These
are obligate or facultative, commensals or symbionts of
insects. The pathogenic action depends on contact and
they infect and kill sucking insect pests such as aphids,
thrips, mealy bugs, whiteflies, scale insects, mosquitoes
and all types of mites [33, 34]. Entomopathogenic fungi
are promising microbial biopesticides that have a multi-
plicity of mechanisms for pathogenesis. They belong to
12 classes within six phyla and belong to four major
groups; Laboulbeniales, Pyrenomycetes, Hyphomycetes
and Zygomycetes. Some of the most widely used
species include Beauveria bassiana, Metarhizium anisopilae,
Nomuraea rileyi, Paecilomyces farinosus and Verticillium lecanii.
Many of them have been commercialized globally (Table 1).
These fungi attack the host via the integument or gut
epithelium (Figure 2) and establish their conidia in the
joints and the integument [35]. Some species such as
B. bassiana and M. anisipoliae cause muscardine insect dis-
ease and after killing the host, cadavers become mum-
mified or covered by mycelial growth [36]. Some fungi,
primarily streptomycetes, also produce toxins that act
against insects [37]. About 50 such compounds have been
reported as active against various insect species belonging
to Lepidoptera, Homoptera, Coleoptera, Orthoptera and
mites [38]. The most active toxins are actinomycin A,
cycloheximide and novobiocin. Spinosyns are commer-
cially available biopesticidal compounds that were origin-
ally isolated from the actinomycete Saccharopolyspora
spinosa [39] and are active against dipterans, hymeno-
pterans, siphonaterans and thysanopterans but are less
active against coleopterans, aphids and nematodes [39].
Nematode Biopesticides
Another group of microorganisms that can control
pests is the entomopathogenic nematodes, which control
weevils, gnats, white grubs and various species of the
Sesiidae family [40-43]. These fascinating organisms
suppress insects in cryptic habitats (such as soil-borne
pests and stem borers). Commonly used nematodes in
pest management belong to the genera Steinernema
and Heterorhabditis, which attack the hosts as infective
juveniles (IJs) [44, 45]. IJs are free-living organisms, which
enter the hosts through mouth, anus, spiracles or cuticle
(Figure 2). They are able to release their bacterial sym-
bionts in to the haemocoel of hosts, killing the host within
24-48 h [46]. The nematodes can complete up to three
generations within the host, after which the IJs leave the
cadaver to find the new hosts [44]. Entomopathogenic
nematodes (EPN) can be mass-produced in vivo and in vitro
in solid media or liquid fermentation [47-49]. Nematodes
that have been successfully produced in fermenters
(7500-80 000-litre capacity) include Steinernema carpo-
capsae, S. riobrave, Steinernema glaseri, Steinernema scap-
terisci, Heterorhabditis bacteriophora and Heterorhabditis
megidis, with a yield capacity up to 250 000 IJs /ml [49, 50].
 6 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources

/
2.5 days

RP Nil
Spore discharge
41111.
Inoculation

Time to kill 3.3 days

Spore formation

0.6 day\ N Death

*lb AL
0.9 days
1.2 days

Insect covered with mycelium Mycelium formation

Saprophytic phase Pathogenic phase

Beauveria bassiana targeting coffee berry borer

1. Infective juveniles (Ijs) search for insect host

2. Us penetrate the host cavity via mouth, anus, spiracles or intersegmental membranes
3. IJs release symbiotic bacteria (e.g.., Xenorhabdus, Photorahabdus) from their gut in host blood
4. Multiplying bacteria cause septicemia and kill the host
5. Nematodes feed on multiplying bacteria and mature into adults
6. Nematodes reproduce and emerge as infective juveniles (IJs) from the host cadaver

Figure 2 (Cont.)

h ttp://www.cabi.org/cabreviews

61.
2 4
1. Larva eats Bt spore having toxic crystal protein
2. High pH in midgut dissolves crystal protein and interact
with epithelial cell receptors
3. Crystal proteins create pores
4. Host organism dies from the invasion
Figure 2 Cartoon representation of effects of a bacterial, viral, fungal and nematode type of microorganisms. (NPV life
cycle source http://en.wikipedia.org/wiki/File:Npv-life_cycle.jpg)
The use of nematodes is done using a curative rather
than prophylactic approach [42], for instance, as demon-
strated in the case of Synanthedon exitiosa, using S. carpo-
capsae and H. bacteriophora nematode species to induce
field suppression of the pest in a curative manner [51];
1 50 000- 300 000 Us/tree were used three times during
September and October for three consecutive years
in order to obtain as much control as was achieved
with chemical pesticides. Some commercial products are
available based on Steinernema and Heterorhabditis nema-
tode formulations (Table 1). However, extensive studies
are required to optimize application parameters and
develop efficient strains to achieve significant control of
pests through nematodes.
Protozoan Biopesticides
Although they infect a wide range of pests naturally and
induce chronic and debilitating effects that reduce the
target pest populations, the use of protozoan pathogens as
biopesticide agents has not been very successful. Protozoa
are taxonomically subdivided into several phyla, some of
which contain entomogenous species. Microsporan pro-
tozoans have been investigated extensively as possible
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Opender Koul 7
O Insect feeding on virus - contaminated fn I lege Virus
Occlusion body
CIO$O up Of CiCrliSiOn bO(firpS 01E10
Nucleus
O Lumen of digestive 11,nt taikalineommions) Cytoplasm
Virus particles being released from 08s and 1.1 e occel
attaching to brush border at gut cells Gut I
O liepliCalkin Of virus in insect cell Plant
NPV life cycle
components of integrated pest management programmes.
Microsporidia are ubiquitous, obligatory intracellular
parasites that are disease agents for several insect species.
Two genera, Nosema and Vairimorpha, have some potential
as they attack lepidopteran and orthopteran insects and
seem to kill hoppers more than any other insect [52].
A study of Nosema pyrausta, a microsporidium infecting
the European corn borer, Ostrinia nubilalis, suggests that in
a horizontal transmission, a spore is eaten by a European
corn borer larva, which germinates in the midgut,
extrudes a polar filament and injects sporaplasm into a
midgut cell. The sporaplasm reproduces and then forms
more spores, which can infect other tissues. Spores in
infected midgut cells are sloughed into the gut lumen and
are eliminated along with faeces to the maize plant. These
spores remain viable and are consumed during larval
feeding so that the infection cycle is repeated in midgut
cells of the new host. If a female larva is infected, Nosema
is passed to the filial generation by vertical transmission.
As the infected larva develops through to an adult the
ovarial tissue and developing oocytes become infected
with N. pyrausta. The embryo is infected within the yolk
and when larvae hatch, they are infected with N. pyrausta.
Both horizontal and vertical transmissions maintain
N. pyrausta in natural populations of European corn borer.
Table 1 Commercial microbial biopesticides developed so far (*products discontinued; **products having EPA experimental use permit) 03

Micro-organism Target pest Action Brand name Producer

Bacteria
A. radiobacter Crown galls Antagonist Galltrol-A AgBioChem
Dygall Agbioresearch Ltd
Norbac 84-C* New BioProducts
Nogall Becker Underwood
B. popilliae Larvae of various beetles Stomach poison Doom Fairfax Biological
Japademic
B. pumilus Effective against rust, downy and Fungicide Ballad Agraquest Inc.
powdery mildews Sonata AS
Yield Shield Gustafson LLC
B. sphaericus Mosquitoes Stomach poison VectoLex Valent Biosciences
B. subtilis Effective against root rot caused by Fungicide and antagonist Serenade Agra Quest, Inc.
Rhizoctonia, Fusarium, Alternaria, Epic Gustafson, Inc.
Aspergillus and Pythium. Also Kodiak
effective against some foliar MBI 600
diseases Companion** Growth Products
Cillus Green Biotech, Korea
Green-all G
Hi Stick N/T** Becker Underwood
Subtilex
B. subtilis FZB24 Effective against Rhizoctonia, Fungicide Rhizo-Plus FZB Biotechnik,
Fusarium, Alternaria, Verticillium Rhizo-Plus Konz GmbH
and Streptomyces on vegetables
and ornamental plants
B. thuringiensis var. aizawai Effective against lepidopterans in Stomach poison Florbac Valent BioSciences
vegetables and maize XenTari
Agree Certis
Design*
Turex
Mattch* Ecogen
Solbit Green Biotech, Korea
B. thuringiensis var. galleriae Effective against American bollworm Stomach poison Spicturin ISCB, India
on cotton, tobacco caterpillar on
chillies, leaf folder of rice and
diamondback moth on vegetables
B. thuringiensis var. israelensis Effective against mosquitoes and Stomach poison Bactimos Valent Biosciences
black fly larvae and midges Gnatrol*
Skeetal
VectoBac
Acrobe American Cyanamide
Aquabac Becker Microbial
BMP
Bacticide Biotech Intl
Bactis Caffaro
Bioprotec AEF Global Inc
 BTI granules Clarke Mos. Cont.
Prehatch SG Meridian
Vectocide Nu-Gro Group
Summit Bactimos Summit chemicals
Teknar Valent Biosciences
B. thuringiensis var. kurstaki Effective against most lepidopteran Stomach poison Bactospeine* Solvay Duphar
larvae and some leaf beetles BioBit Valent Biosciences
Dipel
Florbac
Cordalene Agrichem
Bonide Bonide products Inc
Lipel SP Som Phytopharma
BMP 123 Becker Microbial
Biobest-BT Biobest, Belgium
Scutello
Baturad Cequisa Agro
Worm Wipper* Cape Fear Chem
Collapse* Calliope
Foray Valent Biosciences
Biolep
Condor Certis
Costar
Cutlass*
Foil
Lepinox
Crymax Ecogen/Certis
M-Peril*
MVP II Dow Agro Sci.
Raven*
Ecotech Bio
Ecotech Pro Ecogen/AgrEvo
Jackpot
Rapax Ecogen /Intrachem
FonNabit
Bio-Worm Killer Forward Intl
Guardjet Green Light Co
Maatch Mycogen/Kubota
Batik Mycogen
Bactosid K NPP, France
Bactec BT 16 Nu-Gro Group
Bactec BT 32 Plato Industries
Turibel
Insectobiol Probelte S.A.
Soilsery BT Samabiol
Agrobac Sil Sent Inc
Able* Tecomag SRL
Deliver Certis
Delfin
Table 1 (Cont.)
Micro-organism Target pest Action Brand name Producer
Javelin WG
Thuricide*
Vault* Certis/Valent
Larvo-BT* Valent Biosciences
Troy-BT* Troy Bioscience
Ringer BT Verdant Inc
Safer BTK WoodstreamCanada
Halt Wockhardt Ltd
B. thuringiensis var. tenebrionis Effective against coleopteran Stomach poison Ditera Valent Biosciences
beetles on vegetables Novodor
Trident Mycogen
M-Trak
Erwinia amylovora (HrpN harpin Multi-spectrum Insecticide, fungicide and Messenger Eden Biosciences
protein) nematicide
P. cepacia Effective against soil pathogenic Toxic Intercept Soil Tech
fungi
P. chlororaphis Effective against fungal pathogens Seed treatment control Cedomon BioAgri AB
of barley and oats
P. flourescens Effective against P. tolasi on Antibacterial Conquer Mauri Foods
mushrooms and Erwinia on fruit Blight Ban A506 Nu Farm Inc
crops
P. solanacearum Bacterial control in vegetables Antibacterial PSSOL NPP, Korea
P. syringae Effective against post-harvest Antagonist Bio-Save Jet Harvest Solutions
pathogens on apples, pears and
citrus
Pseudomonas + Azospirillum Effective against brown patch and Antagonist BioJet Eco-Soil
dollar spot soil pathogens
Fungi
Altemaria destruens Cuscuta control Herbicide Smolder G Sylvan Bioproducts
Ampelomyces quisqualis Powdery mildew Control and Hyperparasitic Q-Fect Green Biotech, Korea
damping off disease control Green-all
B. bassiana Effective against variety of insects Insect-specific fungal disease Racer BB Agri Life
such as crickets, white grubs, fire inducers Ostrinil NPP, France
ants, flea beetles, whiteflies, plant B roca ri l Futureco BioSci
bugs, grasshoppers, thrips, Mycotrol Emerald Bioagric.
aphids, mites, mosquito larvae Mycotrol-0
and many others Botanigard
Boverin NextBio
Naturalis-L Troy Bioscience
Naturalis-H&G
Naturalis-T&O
Insecticide Trichobass AMC Chem, Spain
Burkholderia cepacia Effective against soil fungal Controls fungi via seed treatment Deny Stine Microbial Products
pathogens
Candida oleophila Effective against postharvest Colonization of diseased tissues Aspire Ecogen
pathogens like Botrytis and
Penicillium
Coniothyrium minitans Effective against Sclerotinia species Mode-of-action not clear Contans WG Prophyta Biologischer GmbH
on canola, sunflower, peanut,
soyabean and vegetables KONI BIOVED Ltd
Fusarium oxysporum Effective against pathogenic Seed treatment and soil Biofox C
(non-pathogenic) Fusarium on basil, carnation, incorporation Fusaclean SIAPA
cyclamen, tomato NPP, France
Gliocladium catenulatum Effective against Pythium, Mode-of-action not clear Primastop Verdera Oy, Finland
Gliocladium spp. Rhizoctona, Botrytis and GlioMix
Didymella species on greenhouse Prestop Fargo, UK
crops
Gliocladium virens Effective against soil pathogens Antagonist Soil Guardl2G Certis LLC
causing damping off and root rot
Hirsutella thompsonii Effective against mites Stimulates premature fungal Mycar* Formerly Abbot
epizootics
Lagenidium giganteum Effective against mosquito larvae Kill through zoospores Laginex* Agra Quest, Inc
and related dipterans
M. anisopilae Effective against range of pests. Disease-causing fungus Bio 1020* Bayer AG
Green Muscle is specific for Bio-Blast EcoScience
locusts and grasshoppers Bio-Path EcoScience
Biotrol FMA* Nutrilite Products
Green Muscle Bio Control Prod.
Metaquino CODECAP
Pacer MA Agri Life
Ticks Insecticide Tick-Ex Earth Biosci. Inc.
Black vine weevil Insecticide Met52 Fargo, UK
Myrothecium vaerrcaria Effective against many nematodes Nematicidal Ditera Valent Biosciences
Paecilomyces fumosoroseus Effective against whiteflies in Hyperparasitic Preferal WG Biobest, Belgium
greenhouse PFR-97** Certis
Paecilomyces lilacinus Effective against nematodes Antagonist Paecil/Bioact Tech. Innovation Corp/Prophyta
Biologischer
Phelbia gigantea Effective against pine and spruce Biofungicide Rotstop Kemira
rust
Phytophthora palmivora Vine strangler Infects via roots De Vine Abbott
Pythium oligandrum Management of fungal pathogens in Seed treatment or soil Polyversum Biopreparaty Ltd., Czech
various crops incorporation
Streptomyces griseoviridis Effective against wilt, seed rot and Antagonist Mycostop Verdera Oy and
stem rot Mycostop Mix Rincon Vitova
Streptomyces lydicus Effective against soil borne diseases Fungicide Actinovate SP Natural Industries Inc
of turf, nursery crops
Talaromyces flavus V117b Effective against fungal pathogens Antagonist Protus WG Peophyta Biolgischer
of tomato, cucumber, strawberry Pflanzenschutz
and rape oilseeds
Trichoderma harzianum Effective against variety of soil Mycoparasitic, Antagonistic Root Shield BioWorks Inc
pathogens and wound pathogens BioTrek 22g Wilbur-Ellis
Supresivit Borregaard, Denmark
Table 1 (Cont.) It)

Micro-organism Target pest Action Brand name Producer

T-22G Bioworks Inc, EU
T-22HB
Trianum P
Binab Bio-lnnovation
Trichodex Makhteshim, Israel
T. harzianum + T. viride Effective against Armillaria and Mycoparasitic Trichopel Agrimm Tech
Botryoshaeria and others Trichojet
Trichodowels
Trichoseal
Trichoderma sp. Supresses root pathogens Mycoparasitic Trichodry AgrimmTechnologies Ltd.,
Trichoflow New Zealand
Trichogrow
Trichopel
Vinevax
Trichoderma viride Effective against rot diseases Mycoparasitic Ecosom TV Agri Life
Tricon Green Max
AgroTech
Trieco Ecosense labs
V. lecanii Effective against other microbes, Antibiotic and insect eating Mealikil VL Agri Life
aphids, whiteflies and thrips fungus Verticillin Koppert, Netherland
Mycotal
Thriptal Tate and Lyle
Vertalec Koppert, Netherlands
Insecticide
Viruses
Granulosis virus Effective against leafroller and Disease causing virus Capex Andermatt
codling moth and other Carposin Agrichem
lepidopterans Cyd-X Certis
CLV LC
Granupom BioBest, Belgium
Madex 3 Andermatt
Virin-EKS NPO Vector
Virin-GYAP
Virosoft CP4 Biotepp Inc
Carpovirusine NPP, France
Indian meal moth Insecticide Nutguard-V AgriVir LLC
NPV for Anagrapha falcifera Effective against lepidopterans Disease-causing virus AfMNVP Certis
NPV for A. gemmatalis Effective against velvetbean Disease-causing virus Multigen EMBRAPA
caterpillar and sugarcane borer Polygen Agroggen
NPV for Autographa calofornica Effective against Alfalfa looper Disease-causing virus VFN80* Certis
Gusano
NPV for H. zea and H. virescens Effective against bollworms Disease-causing virus Biotrol Certis
Gemstar LC
Elcar Novartis
NPV for L. dispar Effective against Gypsy moth Disease-causing virus Dispavirus* CCIP Corp.
 Gypcheck US Forest Service
NPV for Mamestra brassicae Effective against lepidopterans Disease-causing virus Mamestrin NPP/Calliope
Virin-EKS NPOVector
NPV for Neodiprion sertifer, Effective against sawfly larvae Disease-causing virus Abietiv Sylvar Technologies
N. lecontei and N. abietis Leconteivirus Canadian Forestry Ser Kemira
Monisarmiovirus Oxford Virology
Virox
NPV for Orgyia pseudotsugata Effective against Insecticide Virtuss WP Terra Nostra, Canada
Douglas-fir tussock moth
NPV for Spodoptera exigua Effective against beet and lesser Disease causing virus Ness-A Applied Chemical
army worms, pig weed caterpillar Ness-E
and mottled willow moth Otienem-S Ecogen
Spod-X LC Certis
Gemstar
NPV for Syngrapha falcif Effective against Helicoverpa and Larval disease causing virus NPVSf Certis
Cydia spp.
Nematodes
H. bacteriophora Effective against many lepidopteran Entomopathogen Cruiser* Ecogen
larvae, turf and Japanese beetles Heteromask BioLogic
and soil insects Nema-BIT BIT
Nema-top/-green e-nema
Lawn Patrol Hydro-Gardens
Grubstake Integrated Biocontrol Systems
Larvanem Koppert
Terranem
S. glaseri Effective against root weevils, Entomopathogen BioSafe WG SDS Biotech K.K.
cutworms, fleas, borers and
fungal gnats
H. megidis Effective against black vine weevils Entomopathogen Larvanem Koppert Bio Syst.
and soil insects Dickmaulrussler- Andermatt Biocontrol AG
nematoden
P. hermaphrodita Effective against slugs Slug eating nematode Nemaslug Becker Underwood Inc
S. carpocapsae Effective against black vine weevils, Entomopathogen Bio-Safe-N Certis
strawberry root weevils, Biovector 25
cutworms, cranberry girdler and Savior WG
termites Ecomask BioLogic
Exhibit SC-WDG Novartis BCM
Hortscan BioLogic
Guardian IPM labs/Praxis
Scanmask ARBICO
Termask BioLogic
Millenium Certis
Nematac C Becker Underwood
No Flea Integrated Biocontrol Systems
S. feltiae Effective against vine weevils, Entomopathogen Entonem Koppert
fungus gnats, sciarid flies and soil Exhibit SF-WDG Novartis BCM
insects Nemasys Becker Underwood
Nema-plus e-nema GmbH
14 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
N. pyrausta suppresses populations of European corn
Co
E borer by reducing oviposition, percentage hatch and
a)
Co
survival of infected neonate larvae [53].
(/) The only protozoan registered for use as a biopesticide
0 is the microsporidian, Nosema locustae, which infects
O
grasshoppers (Table 1). This organism is most effective
O when ingested by nymphal stages of grasshoppers and
-(/) 0 a)
o
-0
kills them within three to 6 weeks post-infection [53].
However, not all infected grasshoppers are killed by this
U -ro'
z o_ ir) .° 0 6).T protozoan infection.
-o _o o_-cs -I -t
0
0 .cb<ocoo:10

Microbial Products in Biopesticides

CD:'

-0 1.0 CO
1.0
CD CO (/) 2 In addition to the proteinaceous toxins, microorganisms
ESE
_E
cf) co "6 8 (b)
:-as o0_ are also known to produce anti-pest chemical com-
a) E a) To' E z t',,, '5 -r co 0
c
a)
.

Lo
c
mz 0 a)
ca3
cco-0..,> o
> E of CD
pounds. Fermentation broths provide a readily screenable
. 0 iZ (1) C.) .0 a) a) TD a) source of bioactivity against organisms or targets of
0 (/) HXZU)0000Z
.A= I

ZU)
agricultural interest. Antinsectan compounds derived
from nonfilamentous bacteria (e.g., aminolevulinic acid,
thiolutin, thuringiensin, xenorhabdins); actinomycetes and
a) some fungi (e.g., actinomycin A, aplasmomycin, avermec-
a) tins, citromycin, milbemycins, nikkomycin, piericidins,
4:2
spinosyns, various cyclic peptides, etc.) are well known as
a) 0 toxins, growth inhibitors, antifeedants and physiological
0) _c disrupters against a variety of pests [37, 54, 55]. Some of
O
_c
O 0N -csaS these compounds have been commercialized, such as
oci avermectins and spinosyns.
E O
O O0 a) Some transgenic crops can be considered among
wd" Co
microbially based products. Since 1996, more than
200 million ha of land has been planted with Bacillus-
thuringiensis-based (Bt) genetically engineered crops [56].
as

in
While 29 countries planted commercialized biotech crops
a)
0_ in 2010, an additional 32 countries, have granted regu-
0_
O latory approval for biotech crops for import of food
Co
Co
and feed use and release into the environment since
as
0) 1996. Varieties of these crops produce 18 different
Co combinations of 11 B. thuringiensis toxins, which kill lepi-
dopteran and coleopteran insects. Bt cotton and maize
0)
as have been successful and other crops such as transgenic
0
a)
a) -4-;
> rice, soyabean and rapeseed are making some headway.
a) U
0) Commercial growing was reported in 2009 of smaller
w amounts of genetically modified (GM) sugar beet, papaya,
squash (zucchini), sweet pepper, tomato, petunia, carna-
tions, rose and poplar [57]. Recently, some research and
development has been targeted to enhance crops that are
locally important in developing countries, such as insect-
resistant cowpea for Africa [58] and insect-resistant
brinjal (eggplant) for India [59]. The European Commis-
sion has recently approved Annflora: a GM potato devel-
oped by German chemical company BASF, which is the
first GM crop to be approved for cultivation in the EU for
12 years, after Monsanto's MON 810 maize, which is
engineered to be resistant to the European corn borer
caterpillar, was licensed in 1998 [60].

http://www.cabi.orgicabreviews

Genetic Improvement
Bacteria
The genetic improvement of microbial pathogens aims to
make them more effective by increasing their rate of
reproduction, speed of transmission and infective ability
or increasing the quantity of toxin produced. For example,
genetic transformation of B. thuringiensis has produced
a strain that displays insecticidal activity against both
coleopteran and lepidopteran insects [61]. The activity of
B. thuringiensis on the crop foliage or applications via
soil can also be enhanced by genetic manipulation. For
instance, B. thuringiensis crystal proteins of the Cry34 and
Cry35 classes function as binary toxins showing activity on
the western corn rootworm, Diabrotica virgifera virgifera.
Cry34A/Cry35A pairs are more active than the Cry34B/
Cry35B pairs. The binary Cry34/Cry35 B. thuringiensis
crystal proteins are closely related to each other, are
environmentally ubiquitous and share sequence simila-
rities consistent with activity through membrane disrup-
tion in target organisms. Modified Cry35 proteins whose
segments, domains and motifs have been exchanged with
other proteins to enhance insecticidal activity can provide
excellent control of plant pests and rootworms [62].
Similarly, Cry8Bb1 toxin polypeptide from B. thuringiensis
has been engineered to contain a proteolytic protection
site, which makes it insensitive to a plant protease, helping
to protect the toxin from any proteolytic inactivation.
Modified Cry8Bb1 has been used for controlling corn
rootworms, wireworms, boll weevils, Colorado potato
beetles and the alfalfa weevils [63].
A recent study shows that B. cereus group genomes
have a Bacillus enhancin-like (bel) gene, which has potential
to increase the insecticidal activity of B. thuringiensis-based
biopesticides and transgenic crops based on B. thuringiensis
genes [64]. Bel genes encode peptides, which have
20-30% similarity with viral enhancin protein. These
proteins are known to enhance viral infections as they
degrade the peritrophic matrix of insect midguts. The
combination of Bel and Cry1Ac increased the mortality
rate 2.2-fold [64].
Viruses
Use of recombinant baculovirus technology has a poten-
tial to produce economical substitutes. Recombinant
baculoviruses (vEV-Tox34) expressing the gene Tox-34
from a mite, Pyemotes tritici, increased the speed of kill
of the corn earworm, Helicoverpa zea [65]. Similarly,
two genetically enhanced isolates of the Autographa cali-
fomica nuclear polyhedrosis virus (AcMNPV) expressing
insect-specific neurotoxin genes from the spiders Diguetia
canities and Tegenaria agrestis (designated vAcTaITX-1 and
vAcDTX9.2, respectively) have been evaluated for their
commercial potential against lepidopteran insects. While
http://www.cabi.org/cabreviews
Opender Koul 15
vAcTaITX-1 kills faster than vAcDTX9.2, the latter is a
faster feeding deterrent, suggesting that it would be more
useful in reducing crop damage [66]. However, developing
cost-effective methods for producing recombinant BVs is
very challenging because DNA preparations from these
viruses and their transfection are very labour intensive
and time consuming.
Lymantria dispar multicapsid nucleopolyhedrovirus
(LdMNPV) is used on a limited basis as a gypsy moth
(L. dispar) control agent. In an effort to improve the effi-
cacy (i.e., killing speed) of the LdMNPV, a recombinant
viral strain (vEGT-) that does not produce the enzyme
ecdysteroid UDP-glucosyltransferase (EGT) was devel-
oped. LT50 values of fifth-instar larvae infected with vEGT-
were 33% lower when compared with larvae infected
with a wild virus strain. In addition, Buthus eupeus
insect toxin-1, the Manduca sexta diuretic hormone, the
B. thuringiensis ssp. kurstaki HD-73 delta-endotoxin, the
Heliothis virescens juvenile hormone esterase, the P. tritici
TxP-I toxin, Androctonus australis neurotoxin, Doi m V gene
and T-urf 13 genes have been inserted into BVs for the
purpose of developing viral pesticides [67].
Entomopathogenic Nematodes
In the case of entomopathogenic nematodes, artificial
selection has been successful in increasing infectivity and
nematicide resistance [68]. The strain selection has shown
a gain of fitness with regard to host penetration and
reproductive potential. The recent discovery that maize
roots damaged by the western corn rootworm emit a key
attractant for insect-killing nematodes has opened the
way to explore whether a selection strategy can improve
the control of root pests [69]. Salame et al. [70] bred
a heterogeneous population of Steinernema feltiae for
desiccation tolerance and host-seeking ability after 10 to
25 selection cycles. However, artificial selection for one
trait may come at a cost for other important traits such as
infectiousness, establishment and/or persistence in the
field. Using information from the sequenced genomes of
EPN may enable the production of GM nematodes with
higher storage stability, higher resistance to environ-
mental stresses and higher biological control potential to
be developed in the near future [71-73].
Entomopathogenic Fungi
Two commonly used entomopathogenic fungi, Metarhi-
zium anisopliae and B. bassiana have been extensively
studied for elucidation of pathogenic processes and
manipulation of the genes of the pathogens to improve
biocontrol performance [74]. Additional copies of the
gene encoding the regulated cuticle-degrading protease
Pr1 were inserted into the genome of M. anisopliae and
overexpressed. The resultant strain reduced survival time
16 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
Box 3 Bacterial Biopesticides: A Case Study
Use of B. thuringiensis bacteria for pest management has been
going on for decades. Many studies have come to the fore in
terms of the development of effective strains, formulations,
and subsequently the isolation of Cry proteins was achieved
which ultimately lead to the development of transgenics.
However, the case study presented here is different and
related to the discovery and commercialization of a microbial
biopesticide for a grass grub, C. zealandica based on the
bacterium S. entomophila [139]. These bacteria were first
obtained from sick grubs, cultured and discovered to cause
amber disease in insects. Bacterial septicaemia is accompanied
by a rapid breakdown of the cadaver and release of bacteria
back into soil. The first simple identifications established the
bacteria as non-spore-forming members of the genus Serratia
(Enterobacteriaceae). Subsequently it was established that
S. entomophila and S. proteamaculans bacteria, both of which
could occur in pathogenic and nonpathogenic forms, popu-
lated New Zealand pasture soils. Unlike other soil bacteria,
Serratia can grow on media rich in thalium salts, and caprylate
thallous agar provides an excellent selective medium
for isolation [140]. To develop these bacteria as biopesticides
in tobacco hornworm (M. sexta) by 25% as compared with
the parent wild-type strain [75]. The remarkable extent to
which virulence can be increased is shown in the case of
the scorpion toxin (AaIT) expressed in the M. anisopliae
strain ARSEF 549. The modified fungus gave the same
mortality rates in M. sexta at 22-fold lower spore doses
than the wild type, and survival times at some doses were
reduced by 40% [76]. Similar results have been with
mosquitoes with a 9-fold reduction in LC50 and coffee
berry borer beetle with a 16-fold reduction in LC50 [77].
Production and Development
Some microbial biopesticides are easy to produce and
develop and can be manufactured using simple and in-
expensive technologies. The BVs and EPN can be pro-
duced in vivo in insects and entomopathogenic fungi, such
as Beauveria and Metarhizium, are produced on grains.
Such simple technologies are useful for developing
countries where a substantial demand exists for local
production and distribution at the farmers' level. How-
ever, production methods are only one aspect of
the development of a new microbial biopesticide [78],
and one has to solve potential problems associated
with contamination, formulation potency, attenuation of
pesticidal activity and shelf life. All these aspects require
equipment, expertise, material and capital. As such, small
entrepreneurs, particularly in the developing world are
often not able to meet these requirements and to some
extent, a similar situation persists in small production
facilities in the developed world [79-82]. It is estimated
that to develop a single product costs >US$25 million
http://www.cabi.org/cabreviews
is interesting, because they can be cultured through in vitro
fermentation and can be applied back to soil causing disease
[141]. In the 1980s, Monsanto initiated a programme for
biopesticide development with New Zealand group led by
Murray Willocks to develop a S. entomophila as the product
InvadeTM, which became Monsanto's first and probably
only biopesticide launched on to the market [142] where
only 1 litre of fermenter product was required to treat a
hectare of pasture. Invade Tm was used for several hectares
annually, but a new approach was required to gain greater
market acceptance and key was to develop stable formula-
tions. It was in 2001 that Von Johnson developed a new
flowable granular product with long shelf life in ambient
conditions [143]. Encoate Ltd. and Balance Agri-nutrients
Ltd. in New Zealand have now developed the new
formulations in collaboration with AgResearch, Lincon. The
product BioshieldTM is produced on large scale in the form of
granules (10 tonnes/h). It is now known that Serratia spp.
are capable of holding a range of insect toxins, which may
provide important opportunities for the control of other
pests [139].
from discovery to formulation development in order to
reach a farmer for application.
In the microbial biopesticides field, the major emphasis
has been on Bacillus thuringiensis. Frankenhuyzen [83] has
recently documented that to date 125 of the 174 holotype
known toxins have been tested against 163 test species.
However, the products are classified according to their
formulation. These formulations are emulsions, encapsu-
lations and granules (for agriculture and forestry); wet-
table powders (for gardens); and briquettes (for aquatic
systems). There are now many formulations based on
spore-crystal complexes, which need to be ingested by
the pests for toxic action. The spore-crystal complexes
are required to be carried by suitable excipients that
would protect the spore crystal and at the same should be
palatable to the insect, i.e. with increased feeding pre-
ference. A good example of such a formulation is of Cry
protein from B. thuringiensis with an attractant glycopro-
tein [84] for killing fire ants. Some designs are also known
for beet armyworm, Spodoptera exigua [85]. However, a
classical case study is the discovery and commercialization
of a bacterial product for a grass grub, Costelytra zealandica
based on the bacterium Serratia entomophila (see Box 3).
Many biodegradable materials have been used to prepare
formulations, including liquid or solid carriers, surfactants,
adjuvants, adherents, dispersants, stabilizers, moisturizers,
attractants and protective agents [86]. Long shelf life
and reliable efficacy, which are affected by moisture, are
the two basic impediments for commercialization of a
bacterial biopesticide and strategies to develop delivery
materials with dynamic vapour sorption properties have
been worked out in a recent study where three bio-
pesticide delivery systems, THE -G, PEC-G and PESTA,

Box 4 Fungal Biopesticides: A Case Study
Locusts and grasshoppers are extremely damaging pests in
various parts of the world. The largest locust swarm was
reported in Kenya in 1954, covered more than 1000 km2,
contained 40 000 million insects, and weighed 80000 tonnes.
One tonne of locusts eats as much food in one day as about
2500 people [144]. Similarly, grasshoppers do more crop
damage on an average. The main control measure against
locusts and grasshoppers has been broad-spectrum pesti-
cides. For instance, between 1986 and 1989, donors
and national governments spent US$200 million spraying
10 million ha with 15 million litres of the broad spectrum
insecticides fenitrothion and malathion [145]; the pesticides
having hazardous environmental impacts. In view of such
hazards, the US Agency for International Development
(USAID) began supporting a programme to develop a bio-
pesticide against locusts based on the entomopathogenic
fungus M. anisopliae var. acridum. The LUBILOSA (Lutte Bio-
logique contre les Locustes et Sauteriaux) project was set up in
1989 to develop a biological means of controlling locusts and
grasshoppers. At the beginning, the project involved the CABI
Bioscience in the UK, IITA, and Departement de Formation
en Protection de Vegetaux (DFPV) in Niger. CABI has man-
aged the project but the technical project leader has been an
IITA employee based in Benin since 1992, when LUBILOSA
began small-scale field trials. By the end of Phase 3, the
number of collaborators had increased to include Comite
were analysed by dynamic vapour sorption analysis. The
objective of this study was to demonstrate the moisture
sorption profile of each system in air at 25°C and a
relative humidity (RH) ranging from 0 to 90%. These
studies have revealed that moisture loss retards the
activity in the range of 2.3-3.4 times [87], thus suggesting
the implications relative to moisture distribution.
In the case of fungal biopesticides, a system for mass
production of conidia has been standardized after evalu-
ating different solid matrices such as rice, wheat bran and
mijo grains, contained in both, high-density polyethylene
bags and aluminium trays, supplemented with different
organic nitrogen sources and inoculated with different
inoculum types [88]. Once the matrices are established,
where the greatest conidia production per gram of sub-
strate is obtained, the conidia are separated and used as
an active starter for elaborating the biopesticide proto-
types. Recently, a complex coacervate formulation was
developed for Colletotrichum truncatum, a bioherbicidal
fungus against scentless chamomile, and tested in the
greenhouse. A two-step process was developed to for-
mulate C. truncatum conidia. Firstly, an invert emulsion
preparation of C. truncatum conidia in non-refined vege-
table oil with the aid of a surfactant was prepared, fol-
lowed by encapsulating the C. truncatum conidia invert
emulsion by complex coacervation. Formulation ingre-
dients included non-refined vegetable oils, surfactants,
proteins and carbohydrates. Most formulation ingredients
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Opender Koul 17
Inter-Etat de Lutte contre la Secheresse au Sahel (CILSS),
Deutsche Gesellschaft fur Technische Zusammenarbeit
(GTZ) in Germany, and two private companies, Biological
Control Products (BCP) in South Africa and Natural Plant
Protection (NPP) in France.
In the first 10 years, the LUBILOSA project spent US$15
million and produced an environmentally benign alternative to
chemical pesticides. Demonstration trials and farmer partici-
patory trials have been conducted in most Sahelian countries
in collaboration with the national programmes [146].
A project in Australia has used LUBILOSA research data to
develop a biopesticide against Australian locusts [147]. The
product kills 80% of insects within 1 to 3 weeks. A company is
licensed to manufacture the biopesticide, which has been
registered in South Africa under the name Green Muscle®.
This consortium of donors has recently funded a fourth phase
to "steward" the LUBILOSA biopesticide to higher adoption
rates and greater impact.
As such, LUBILOSA may well be a template for much more
of the activities of the Consultative Group on International
Agricultural Research (CGIAR) in the future. Hence, an
analysis of the impacts that LUBILOSA has had, could
have in the future, and how this impact has and can be
achieved, can teach us a great deal about public-private
partnerships and the management of impact-focused research
[148].
considered and tested in this study were compatible with
C. truncatum, with no significant reduction in conidial
germination and mycelial growth. The surfactant soya
lecithin promoted the greatest retention of C. truncatum
conidia (88%) in the invert emulsion. In greenhouse
studies, scentless chamomile disease was controlled sig-
nificantly [89]. This example implies that fungal microbial
biopesticides could be very useful in field situations if
appropriate formulations and specific delivery systems
are developed. However, specific example of successful
fungi-based product is the LUBILOSA programme (see
Box 4).
The use of wild-type biopesticides in terms of regis-
tration could be more appropriate as they will require a
demonstration of efficacy and safety issues will be of less
concern. There is evidence to show the potential of wild-
type BVs [90, 91]. For instance, process patent is available
for production of BVs via fermentation in Australia [92],
which targets the Helicoverpa pest species and accounts
for a US$3.2 billion per annum market. Production costs
suggested allows to target Helicoverpa pest species in
areas where this pest is resistant to most low-cost
chemical options (15 $/ha), and where only more expen-
sive chemicals are in use (30-50 $/ha). These studies
suggest that wild-type products with improved yields can
compete on cost alone in all markets, including extensive
markets in India and China. Therefore, bioreactor-based
production of BVs requires more focus, which may be
18 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
Box 5 Viral biopesticides: A case study
Control of the velvetbean caterpillar, A. gemmatalis, in soya-
bean in Brazil is an interesting case study where one can take
an advantage of a naturally occurring nucleopolyhedrosis virus
(AgMNVP) against a pest. This virus is currently used on
approximately two million hectares of soyabeans in Brazil and
is one of the largest programmes worldwide for the use of a
viral pathogen to control a pest of a single crop [149].
Although implementation of the programme began in the
1982/83 season, it gained momentum with the development
of a wettable powder formulation in 1986, which was then
taken up by private companies for commercial production;
use of AgMNVP reached 1.5 million ha in 1995. Field pro-
duction of the virus became a big business in Brazil, involving
different persons and small companies specialized in selling
AgMNPV-killed caterpillars [150]. A breakthrough in com-
mercial pilot laboratory production occurred at Embrapa
Soybean in Londrina in 2002. In the production system of the
virus, eggs are obtained daily in adult oviposition rooms, and
larvae reared in separate rooms up to fourth instar in 500 ml
Box 6 Nematode biopesticides: A case study
Molluscs are pests for which few biological control agents are
available. Therefore, this is a case of a successful commercial
development of a novel species of nematode with specific
activity against slugs. A nematode, P. hermaphrodita has been
isolated from a destructive grey field slug species D. reticu-
latum [151], which develops a very characteristic swelling in
the rear half of their mantle at an early stage of infection, and
following death, many large nematodes of 3 mm size can be
seen feeding on the cadaver. This nematode has a curious
ability to adopt necromenic life cycle in larger slugs, in which
the Us enter the slug's body cavity and remain dormant there
without doing harm until the slug dies, when the juveniles
develop and reproduce, feeding on the cadaver. When the
nematode enters smaller slugs, it develops directly, causing
disease and death of the host. An interesting thing about
cost-effective and high yielding. However, control of the
velvetbean caterpillar, Anticarsia gemmatalis in soyabean in
Brazil is an interesting case-study based on laboratory
production where one can take an advantage of a naturally
occurring nucleopolyhedrosis virus (AgMNVP) against
pest (see Box 5).
EPN can be commercially mass-produced in vivo or
in vitro, in solid or in liquid culture, each system having its
own advantages and disadvantages relative to costs of
production, investments, quality of products and tech-
nology. One of the good examples is a successful com-
mercial development of a novel species of nematode
with specific activity against slugs where a nematode,
Phasmarhabditis hermaphrodita has been isolated from a
destructive grey field slug species Deroceras reticulatum
(see Box 6). However, there are many challenges in
making production more reliable and economical [93] and
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cardboard cups containing an insect diet. Daily 3% of the
larvae are transferred to plastic trays with diet and vermi-
culture to obtain pupae and maintain the insect colony. The
rest (97%) of the fourthh instars are taken to the virus pro-
duction laboratory, where they are transferred to plastic
trays containing AgMNVP- treated diet. Seven days later, dead
larvae are collected into plastic bags with a modified hand
vacuum cleaner. The larvae are then taken to a storage room
for further processing and formulation of the product. This
commercial laboratory production started in the end of 2004
and produces 800 000-1 000 000 larvae/day, resulting in
agMNVP to treat 1.8-2.0 million ha/year [149]. It is expected
that AgMNVP use may reach to 4.0 million ha/year by 2012.
This programme in Brazil has been successful because of its
implementation of a soyabean IPM programme, proactive
activities of extension units, high virulence of pathogen to
the host and efficient horizontal transmission, continued
exposure of pest to AgMNVP and ability to produce large
quantities of virus under field conditions at a very low cost.
nematodes such as EPN is their mutalistic symbiosis with
Gram-negative entomopathogenic bacteria such as Xenor-
habdus and Photorhabdus, but this is not true in case of
P. hermaphrodita. This nematode is capable of growth on a
wide range of bacteria, but nematodes grown on different
bacteria differ dramatically in virulence. The reason for this is
unknown [152]. However, the use of a bacterium, Moraxella
osloensis that produced consistently pathogenic nematodes
for mass production in the variety of field experiments has
been highly significant and economically viable. The product
based on this nematode is now making a transition from being
a garden and protected crop treatment to being used in field
vegetables [153]. The very famous product Nemaslug is sold
in UK and each commercial packet contains 12 million dauer
juveniles of P. hermaphrodita.
process parameters require more research, particularly
the ones that influence the bacteria as well as the nema-
todes in liquid cultures. Another significant factor is phase
variation of the symbiotic bacteria, shifting from primary
to secondary forms that lead to unpredictable yields [94].
As of today, a huge number of Us is required for pest
management within a location; if these numbers could be
decreased, market opportunities for EPNs will definitely
increase.
In terms of the formulation development, the ability to
formulate viruses for application with commonly available
implements makes viruses more attractive as biological
control agents. Commercially, dried formulations have an
advantage over liquid formulations for storage and hand-
ling. However, dry formulations have constraints such as
dustiness, inhalation risk and storage. Lyophilization has
been the most common method for stabilizing viruses but

is an expensive procedure [95]. Another method has been
the encapsulation with cornstarch to prepare granular
formulations. The lignin formulations have demonstrated
extended residual activity in both laboratory and field
experiments [96, 97]. Spray drying [98] has been used for
B. bassiana to microencapsulate conidia of this entomo-
pathogenic fungus at low temperature [99, 100]. In addi-
tion, various polymers are used to prolong the activity
of the conidia and improve their shelf life. The best-
encapsulated product, composed of 10% dextrin, 10%
skimmed milk and 5% polyvinylpyrrolidone K90 as the
coating material, had a shelf life of 6 months at 4°C [101].
However, ingredient selection, processing techniques and
moisture content still hinder the development and pro-
duction processes of virus-based microbial biopesticides.
Some delivery systems through drip irrigation systems
have also been studied recently. The suspendible entomo-
pathogenic fungi and EPN evaluated through drip lines are
a viable alternative for application of water-soluble and
insoluble materials; however, the discharge rates need
to be determined for uniformity of the delivery [102].
Nematode formulations, however, need to be tailored and
require the studies in physiological chemistry of nematode
and its ecology and behaviour. A recent overview of
nematode-based formulations [103] advocates the use of
many types of formulations, including water-dispersable
granules. However, water-dispersable granular formula-
tions were not successful on a larger scale [104]:
dominant and successful ones were clay- and gel-based
formulations.
Resistance to Microbials
Among the various groups of microbial pathogens,
development of resistance has been most frequently
reported in the case of B. thuringiensis. Within the last few
years, at least 16 insect species have been identified
that exhibit resistance to B. thuringiensis 8-endotoxins
under laboratory conditions and field-evolved resistance
has been documented in noctuids such as Spodoptera
fusca and H. zea [105]. Reports
frugiperda, Busseo la
of development of resistance in field populations of
Plutella xylostella are essentially from the countries where
Bacillus thuringiensis is extensively used, i.e. China, Japan,
Phillippines, Malaysia, India and North America. To avoid
this resistance problem, genetic engineering was con-
sidered as a useful tool where microbial genes from
B. thuringiensis were transferred to plants to produce
transgenics and today we have B. thuringiensis cotton and
B. thuringiensis maize available in 13 and 9 countries,
respectively, grown on 42.1 million ha of land [106]. The
development of such transgenics was seen as a panacea in
terms of microbial control of pests; however, field resis-
tance in H. zea as a result of an increase in the frequency
of resistance alleles is alarming [107]. The field-evolved
insect resistance to B. thuringiensis crops and various
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Opender Koul 19
aspects related to resistance monitoring methods have
been comprehensively reviewed recently [105]; obviously
more prominent in lepidopterans [108, 109]. Factors
associated with field resistance are the failure to use high-
dose B. thuringiensis cultivars and lack of a sufficient refuge.
While implementation of the high-dose/refuge insect
resistance management strategy has been successful in
delaying field resistance to Bt crops [109], gene pyramid-
ing is another approach used to try and address the
emerging resistance problem [110, 111]. Pyramiding
means the stacking of multiple genes so that more than
one toxin is expressed in the transgenic plant. However,
gene-pyramiding needs to be sustainable and no cross-
or multiple resistances should occur. The problem of
developing multiple resistance cannot be summarily
ignored as in the end they would render such strate-
gies ineffective. Asymmetrical cross-resistance between
B. thuringiensis toxins Cry1Ac and Cry2Ab in pink boll-
worm [112] suggests that it is important to incorporate
the potential effects of such cross-resistance in resistance
management plans so as to help to sustain the efficacy
of pyramided B. thuringiensis crops. Current evidence
suggests that gene pyramiding may not be a sustainable
strategy per se; therefore, other management strategies
such as refugia, use of predators and parasitoids and crop
rotation strategies need to be incorporated in the man-
agement plans [110, 112]. Transgenic plants that control
insects via RNA interference are going to be a reality
soon [113, 114], which will broaden further the scope of
transgenics and can help in minimizing the drawbacks of
resistance. Some recent studies have shown that toxin-
binding proteins such as cadherin promote B. thuringiensis
toxicity [115]. These binding proteins facilitate toxin
oligomerization and thus modify the B. thuringiensis toxin,
which can prevent the resistance in comparison with the
standard B. thuringiensis toxins. The studies demonstrate
that the toxicity of B. thuringiensis toxin Cry1Ab is reduced
by cadherin gene silencing with RNA interference in
M. sexta. The toxins that had cadherin deletion mutations
killed cadherin-silenced M. sexta and B.-thuringiensis-
resistant Pectinophora gossypiella [115].
Recently, resistance in a baculovirus in the field has
been found in Europe where Cydia pomnella GV is one
of the main components of the codling moth control.
C. pomonella GV in apple orchards has led to a high degree
of resistance in some populations [116, 117]. This is the
first documented instance of field resistance to a com-
mercially applied baculovirus [118]. Apparently, this is
either the result of the overuse of the product or the
predominant control strategy applied. However, there do
not seem to be any reported examples of field develop-
ment of resistance to entomopathogenic fungi or nema-
todes [119]. However, there is evidence to demonstrate
the existence of natural resistance mechanisms in insects
against fungi [120, 121] and nematodes [122], suggesting
that resistance to these pathogens cannot be summarily
ignored.
20 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
Future Perspectives
Owing to some of the early successes and the continuing
growth of biopesticide market, expectations for the
performance of microbial biopesticides have been high.
However, there are many challenges that will need to be
overcome. Firstly, questions have been raised about the
barriers that research patents place on humanitarian uses
of patented technologies as well as on the conduct and
availability of publicly funded research results [1 23, 1 24].
Secondly, current regulatory guidelines are inadequate,
while information on the uptake of microbial control
strategies must be collated and shared with the rest of
the field. Furthermore, to implement local production
schemes in developing countries, intervention at the
national and international level will be important.
Thirdly, there is also a need to look into the ecological
relevance vis-à-vis the use of microbial biopesticides.
Some recent studies reveal that pattern and impact
of these toxins varies from species to species, depending
on the ecosystem, the route of exposure and the
non-Bt control against which effects are quantified
[125]. As such, the effect of microbial biopesticides
on microbial communities must be carefully monitored
[126].
In fact, there is a need for well-defined selection criteria
and a complete process description for the development
of a microbial pest control product. For a commercial
microbial product, three specific criteria for selection are
required, i.e. toxicity, production efficiency and safety of
the product. That means while screening process toxicity
of the product will be relative to dose rate, mode-of-
action, speed of kill, host range, sensitivity to abiotic fac-
tors and persistence. Secondly, mass production will be
critical criteria and should be a high-yield-oriented pro-
cess. Thirdly, safety of product will be essential in relation
to registration requirements and the costs involved.
An important question, however, is when are microbial
biopesticides appropriate? Generally, scientists and bio-
control companies seem to develop their products
without a well-developed plan, though the approach
should be to develop a product to solve a problem or to
grasp an opportunity. It is essential to make a detailed
characterization like which pest, crop, region, time of
the problem, solutions available, acceptable costs and
market potential [1 27]. If these aspects are considered
and details are provided, a potential microbial product
could be obtained. A good example is the Lubilosa (see
Box 3), which was a problem-solving project. Therefore,
recommended steps to obtain a good microbial pest
control product would be: (i) collection of isolates and
identification of perfect isolate, (ii) laboratory screening
for efficacy, (iii) assessment of production efficiency,
(iv) mode-of-action and toxicological properties, (v) glass-
house trials and (vi) evaluation of efficacy under com-
mercial conditions. If all these factors are considered,
success is inevitable, perseverance to develop such
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products will be rendered less risky, and questions such
as, 'how to walk a tightrope' [128] or 'The long and
winding road - discovery to commercial product: are we
there yet?' [1 29] will be answered.
In order to increase the utility of microbial pathogens
in EBIPM programmes, systematic surveys are required
in different agroecological zones to identify naturally
occurring pathogens. Detailed studies are necessary on
the properties, mode-of-action and pathogenicity of such
organisms. Ecological studies on the dynamics of diseases
in insect populations are necessary because the environ-
mental factors play a significant role in disease outbreaks
and ultimate control of the pests. It is expected that with
the recent advancements in microbial research coupled
with dedicated efforts from extension specialists, farmers,
pest management regulators and the general public,
microbial biopesticides could play a prominent role in
future EBIPM and AWPM programmes. As mentioned
above, structured project plans are required to
achieve the goal. The roadmap to successful development
and commercialization of a microbial pest control
product is amply illustrated in new flow diagrams recently,
which provide the details of various phases' involved
and output information leading to consecutive steps
for decision making and ultimately the market potential
[127].
With respect to the ecology of microbial control
agents, this has been a major concern over the years that
has remained little researched. So far, biotechnology
and genetics has (understandably) driven the progress in
microbial control but there has been negligible interest in
how these organisms have evolved to survive in nature.
For example, the means by which B. thuringiensis survives
in nature have yet to be proven [1 30], and the costs and
benefits of cry gene possession are unclear. It is evident,
however, from the plethora of cry genes that exist that
gene duplication and exchange are commonplace. In fact,
its spore-forming nature makes it uncertain with regard to
how much vegetative existence B. thuringiensis has and so
how much mutation and recombination can take place. It
certainly has a vegetative existence in at least some insect
species, in which it causes pathology through conjugation
[1 31]. An interesting aspect would be to see if the
same lack of association between sequence types and
episomal factors are evident in other environments where
B. thuringiensis can be demonstrated to have a vegetative
existence [1 32]. A recent study [1 33] with seedlings
of clover (Triflorium hybridum) shows colonization by
B. thuringiensis when spores and seeds were co-inoculated
into soil. Both a strain isolated in the vegetative form from
the phylloplane of clover and a laboratory strain were able
to colonize clover to about 3 times higher density when
seeds were sown in sterile soil rather than in non-sterile
soil. A strain lacking the characteristic insecticidal crystal
proteins produced a similar level of colonization over
a 5-week period as the wild-type strain, indicating that
crystal production was not a mitigating factor during

colonization. A small plasmid, pBC16, was transferred
between strains of B. thuringiensis when donor and reci-
pient strains were sprayed in vegetative form onto leaves
of clover and pak choi (Brassica campestris var. chinensis).
The rate of transfer was about 0.1 transconjugants/
recipient and was dependent on the plant species. The
levels of B. thuringiensis that naturally colonized leaves
of pak choi produced negligible levels of mortality in third
instar larvae of Pieris brassicae feeding on the plants.
Considerable multiplication occurred in the excreted
frass but not in the guts of living insects. Spores in the
frass could be a source of recolonization from the soil and
be transferred to other plants. These findings illustrate
a possible cycle, not dependent on insect pathology, by
which B. thuringiensis diversifies and maintains itself in
nature. The majority of research that has been carried out
on B. thuringiensis has related to its insecticidal toxins and
its survival in nature may not always depend on insect
pathology. It can colonize seedlings from spores in the
soil, exchange genetic information on the phylloplane, and
an appreciable multiplication can occur in the frass of
insects that it did not kill [133]. A cycle of transmission
and survival can thus be envisaged. The enigma, however,
is the cost and benefit of crystal protein production in its
ecology, particularly when in competition with non-crystal
protein producing bacteria such as B. cereus. Therefore,
longer-term studies in nature or in microcosms and
their survival in soil and plants in the presence of sus-
ceptible and non-susceptible invertebrates are required
[133].
The mechanism of resistance, specifically for Cry
proteins, is a matter of concern. Recently, a database
consisting of 12519 high-quality sequences have been
developed from the larval gut of European corn borer.
This obviously can provide basis for future research to
develop gut-specific DNA microarrays to analyse the
changes of gene expression in response to B. thuringiensis
protoxins/toxins and the genetic difference(s) between
Bt resistance and susceptible strains [134]. In fact, 52
candidate genes have been identified that may be involved
in Bt toxicity and resistance. For instance, out of selected
genes, five genes with decreased expression and ten
with increased expression in Cry1Ab-resistant strain of
European corn borer may help in identifying the genes
involved in Bt resistance that could provide new leads into
the mechanism of Cry1Ab resistance in these insects
[134].
Commercialization is the final and most difficult step in
the development of a microbial product. The most critical
factors are developmental cost and time to market. Costs
amount to US$14 - 21 million for a new entrepreneur
and the time to market including registration is no less
than 5-7 years. Therefore, to examine all these critical
factors in the successful commercialization of microbial
pest control products is essential in the developmental
process of a product and these critical factors have been
comprehensively discussed recently [127].
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Opender Koul 21
Acknowledgements
This review article contains information gathered from
numerous published resources, and thus I would like to
extend my appreciation to all authors of the references
used in this manuscript.
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