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Bacterias entomopatógenas:
Su utilización en el control de plagas

Ing. Agr. Graciela Beatriz Benintende

Insumos Bacterianos, Procesos y Formulaciones
IMYZA, INTA
benintende.graciela@inta.gob.ar

Over 90 species of naturally occurring, insect-specific (entomopathogenic) bacteria

have been isolated from insects, plants, and the soil, but only a few have been studied
intensively and developed for biological control. Much attention has been given to
Bacillus thuringiensis, B. sphaericus, Paenibacillus popilliae and Serratia entomophila;
species that has been developed as microbial insecticides.

Bacillus thuringiensis - Based Biological Control of Insect Pests

What is Bacillus thuringiensis Berliner (BT or Bt)?

 A rod-shaped (1.0-1.2 by 3-5 micron), gram-positive, facultative anaerobic, spore

forming bacterium.
 A facultative pathogen of some insect species commonly used as a biopesticide
(microbial control agent).
 More than 150 insects belonging to the orders Lepidoptera, Diptera, and
Coleoptera are known to be susceptible to Bt. To be effective, Bt must be eaten
by insects during their feeding stage of development, when they are larvae. Bt is
ineffective against adult insects.
 During sporulation, it produces protein inclusions (parasporal inclusions) adjacent
to the endospore.
 The parasporal inclusions consist of one or more insecticidal proteins in the form
of a crystal or crystal-complex. These insecticidal proteins are commonly known
as Insecticidal Crystal Proteins (ICP) or delta (d) endotoxin. The primary
insecticidal activity of Bt is due to ICP. It is the active ingredient of most (90%) of
the microbial insecticides produced in the world.

Electron Micrograph of a Sporulating Bt Cell

 The endospore, highly resistant to environmental stress, provides a mechanism

for long-term survival of Bt.
 Besides ICP and endospore, some Bt subspecies produce Beta Exotoxin which
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is toxic to all forms of life including humans.

 An insect must have a strongly alkaline mid-gut, specific proteolytic enzymes and
suitable tissue receptor-sites, to be susceptible.
 Bt can be produced easily on solid media or under submerged fermentation
conditions - a key factor in its successful development as a biopesticide.
 Biotechnology and advances in the fermentation industry over the past three
decades have improved the quality of Bt products.
 Bt is key source of genes for transgenic expression to provide pest resistance in
plants.

Natural Habitat

 Plant surfaces - Bt has been found extensively in the phylloplane. Numerous

subspecies have been isolated from coniferous trees, deciduous trees, and
vegetables. It has also been recovered from stored products.
 Soil - spores persist in soil, and vegetative growth occurs when nutrients are
available.
 Insect Hosts - various Bt subspecies have been isolated from dead or dying
insect larvae.

Environmental Fate

For lepidopteran and coleopteran pests, Bt is applied to the surfaces of the plants and
for dipteran pests (mosquitoes and blackflies) applied to their aquatic, larval habitats.
Many Bt insecticides show poor stability under field conditions, and so frequent
reapplication is required.

A. Terrestrial Habitat [Plant Surfaces (Phylloplanes) and Soil] - Coleopteran-active

and Lepidopteran-active subspecies

 Bt ICPs are degraded quickly by solar radiation

 Bt Endospores are inactivated rapidly when exposed to UV radiation
 Half-life: a few hours - 10 days in most agricultural crops.
 Vegetative cells and spores may persist at gradually decreasing concentrations
for weeks, months, or years as a component of the natural microflora.

B. Aquatic Habitats - Dipteran-active subspecies

 Rapid sedimentation in all but the fastest flowing stream

 Spores may persist for at least 22 days in sediments.
 Contact of Bt subspecies israelensis with mud result in an immediate
disappearance of larvicidal activity.

Classification of Bt Subspecies

 Based on the serotype (serological analysis of flagella antigens) supplemented

by morphological and biochemical criteria.
 Over 84 subspecies have been identified; subspecies commonly used in
agriculture/public health.
 Bt subspecies kurstaki (Bt k) - controls various types of lepidopterous insects.
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 Bt subspecies israelensis (Bt i) - effective against mosquitoes, blackflies’ and

some midges.
 Bt subspecies tenebrionis (also formerly as subsp. san diego) (Bt te) - effective
against certain beetle (chrysomelids) species and the boll weevil.
 Bt subspecies japonensis (Bt j) - effective against many species of scarabid
beetles.
 Bt subspecies aizawai (Bt a) - used against wax moth larvae in honeycombs.

Pathogenicity/Toxicity

Toxicity is due to ICP, endospore or Beta Exotoxin.

Insecticidal Crystal Proteins (ICP)

 Also called as delta-endotoxin. They are protoxin (it must be activated before it
has any effect).
 Commonly designated as “Cry” proteins encoded by “cry” genes.
 The genes that encode ICP are mostly on plasmids; each ICP is the product of a
single gene.
 A subspecies can synthesize more that one type of ICP.
 ICPs have various forms (bipyramidal, cuboidal, flate rhomboid, or a composite
with two or more crystal types).
 Size: Protoxin - A large protein of up to 130 kilo Daltons (kDa), Active Toxin - 65
kDa.
 Extremely potent and toxic to target insect larvae at picomole concentrations.
 Most susceptible species belong to the orders Lepidoptera, Diptera, and
Coleoptera.
 Most susceptible insects are killed by ingestion of the crystals alone; a mixture of
spores and crystals are required for a toxic effect in only a small number of
insects.
 A mixture of different Cry toxins results in a synergism of insecticidal activity.
 A mixture of Cry toxins and Bt spores results in a synergism of insecticidal
activity in several insect pests including Indian meal moth, Plodia interpunctella
(due to either additional toxins in the spore coat or the outgrowth of vegetative
cells from ingested spores).
 Highly insoluble in normal digestive system conditions - safe to humans, higher
animals, and most insects.
 It is soluble in reducing conditions of high pH (above pH 9.5) - a condition
commonly found in the mid-gut of lepidopteran larvae. For this reason, Bt is a
highly specific insecticidal agent.
 The midgut of susceptible insects contains the correct pH and enzymes to
transform the crystal into an active toxin.
 Different domains of the ICP are responsible for host susceptibility (receptor
recognition) and toxicity (pore formation).

Endospores

 Endospores are resistant to inactivation by heat and desiccation and persist in

the environment under adverse conditions.
 Provide a mechanism for long-term survival of Bt.
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 Are pathogenic for some insects, particularly when combined with ICP.

Beta Exotoxin

 During vegetative growth, some Bt subspecies (thuringiensis, galleriae, and

darmstadiensis) produce Beta Exotoxin, an ATP analogue, water soluble, heat
stable secondary metabolite.
 Beta-exotoxin is an inhibitor of RNA polymerase and acts competitively with ATP
in various biological processes.
 It is toxic to almost all forms of life including humans. It has broad-spectrum
insecticidal activity.
 Products with Beta Exotoxin are prohibited in the USA. Manufacturing process
includes monitoring to prevent Beta Exotoxin from appearing in products.

Mode of Action

Bioactivity of Bt is dominated by the ICPs and can be summarized in the following

stages:

 Ingestion of spores and ICP by a susceptible insect larva;

 ICP dissolve in the alkaline midgut;
 Activation of the ICP by proteases;
 Irreversible binding of the C-terminal domain of activated ICP to specific
receptors in the midgut cell membrane;
 Insertion of the N-terminal domain of activated toxin in the cell membrane and
formation of pores and channels in the gut membrane, followed by destruction of
epithelial cells and paralysis of the digestive system;
 Spore germination and septicemia;
 Larval death (within an hour to a few days) from starvation or septicemia.

Effects on Insects

 Immediate cessation of feeding and general paralysis of the larval midgut.

 Subsequent symptoms include vomiting, diarrhea, general sluggishness, and
general paralysis.
 Death may follow immediately or in an hour to a few days.
 Sublethal effects may include: reduced feeding, reduced larval and adult
longevity, reduced fecundity, reduced larval and adult body weight.

Using Bt in Integrated Pest Management

A. Host Range

Larvae of the insect orders Lepidoptera, Diptera, and Coleoptera

B. Use Profile

 Bt is the most versatile biopesticide for use in pest management.

 It is used on agricultural crops, harvested crops in storage, ornamentals, bodies
of water, and around the home to control various groups of insects, depending on
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the type of toxin produced by the specific isolate of Bt.

 Currently, over 180 Bt products are registered in USA.
 Bt is primarily a pathogen of insect larval stages. Younger larvae are more
susceptible than older larvae.
 There are different strains or varieties of Bt available that have been selected for
the control of specific insects.
 It is compatible with many pesticides and can be used mixed with chemical
insecticides.

Ecotoxicity/Non-Target Effects

 To date, there is no known mammalian health effect.

 At the label use rates of registered Bt active ingredients, non-target effects are
minimal to nonexistent for birds, and aquatic organisms such as fish and
invertebrates.
 Applications of formulated Bt are not toxic to most beneficial or predator insects.
Treatment of honeycombs with Bt subspecies aizawai does not have a
detrimental effect upon bees, nor on the honey produced. Very high
concentrations (108 spores/ ml sucrose syrup) of Bt subspecies tenebrionis,
which is used against beetles such as the Colorado potato beetle, reduced
longevity of honey bee adults but did not cause disease. Bt applied at rates used
for mosquito control may cause the death of some non-target species. It did not
have negative effects on frogs and salamanders.
 Threatened and Endangered Species: Bt may be a hazard to endangered
species if it is applied to areas where they live. Certain uses of Bt will jeopardize
the continued existence of endangered moths, butterflies, certain beetles, and
some flies (dipterans); effects vary depending on the subspecies of Bt used.

Advantages of Bt

 Bt is essentially non-toxic to humans, pets and wildlife(inhalation or contact with

eyes or open wounds should be avoided). This high margin of safety
recommends its use on food crops or in other sensitive sites where chemical
pesticide use can cause adverse effects.
 There is no waiting period from time of application before re-entering the field.
 Different strains of Bt are class-specific, meaning beneficial or non-target insects
are not harmed.
 The insects that ingest Bt and later die from it, are not considered dangerous to
birds or other animals that may feed on the dead insects.
 Bt is not known to cause injury to plants on which it has been applied and is not
considered harmful to the environment.
 There have been only a few of cases of Bt resistance reported to date; overuse
must be avoided. Relying on any one pesticide can lead to the build up of
resistance in the pest population. Pesticide rotation and cultural control methods
should be used to slow down or eliminate the possibility of resistance
development.
 Bt use can integrate well with other natural controls when trying to control a pest
insect. For example, the use of Bt products in the apple and pear ecosystem
does not cause spider mite problems; mite outbreaks commonly result following
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destruction of their natural enemies by less selective treatments.

Disadvantages of Bt

 Bt acts as a stomach poison; consequently good coverage is essential; Bt must

be eaten to be effective.
 It is only effective against immature stages of Lepidoptera, Diptera, and
Coleoptera feeding on exposed plant surfaces. This limits its usefulness against
insect pests that tunnel into the plants or plant parts. Examples include codling
moth and corn earworm, which are susceptible to Bt but rarely have an
opportunity to eat it in field use.
 It lacks transmission and spreading facilities, it must be applied like a chemical
insecticide.
 Bt does not induce epizootics (disease outbreak) except in cases where insects
are in confined or crowded area such as bee-hives, insect rearing facilities, or
stored grain bins.
 Short residual effect, more frequent applications are required compared with
chemical insecticides. Bt is susceptible to degradation by sunlight and most
formulations persist on foliage less than a week following application. Even
shorter persistence occurs with some of the newer strains (developed for leaf
beetle control), which may become ineffective in about 24 hours.
 The highly specific activity of Bt insecticides might limit their use on crops where
problems with several pests occur, including non-susceptible insects (aphids,
grasshoppers, etc.).
 Bt-based products tend to have a shorter shelf life than other insecticides.
Manufacturers generally indicate reduced effectiveness after two to three years
of storage, with liquid formulations being more perishable than dry formulations.
Shelf-life is greatest when storage conditions are cool, dry, and out of direct
sunlight.

Paenibacillus popilliae and P. lentimorbus

Naturally occurring bacteria that have been mass-produced for the control of Japanese
beetle larvae in turf since the 1940s. Several commercial products are available. The
bacteria, usually applied to the soil, cause “milky disease.” The term comes from the
larva’s pure white appearance when infected with the pathogens.
P. popilliae was the first insect pathogen to be registered in the U.S. as a microbial
control agent.
Milky disease spores may reproduce within the beetle larvae and establish a resident
population capable of causing mortality over several seasons if the soil is sufficiently
warm and moist through the summer months. It may take several seasons for the
disease to control the pest, and it is preferable to treat a broad area to reduce the
impact of immigrating healthy beetles.

Relative effectiveness

The advantages of using commercial preparations of P. popilliae include the very

narrow host range (they are effective against Japanese beetles, only), their complete
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safety for man and other vertebrates, their compatibility with other control agents
including chemical insecticides and insect-pathogenic nematodes, and their
persistence.

The disadvantages include the high cost of production in vivo, the slow rate of action,
the lack of effect on adult Japanese beetles, which also cause obvious and distressing
damage, and the need for large areas to be treated for effect. The narrow host range,
which is environmentally very desirable, is also a disadvantage: managers must
accurately identify the infesting grub species to determine if it is Japanese beetle. If
there are other grubs present, they will not be attacked.

Bacillus sphaericus (Bs)

A bacterium that is used to control disease transmitting mosquitoes, especially to kill

Culex mosquito larvae in highly organic water. Several different varieties of Bs have
been found in soil and aquatic habitats. The different varieties of Bs can be divided into
five genetically distinct groups. One of the groups includes varieties effective against the
larvae of pathogenic mosquitoes. During sporulation, these varieties produce parasporal
bodies that contain larvicidal toxins. Following ingestion of the parasporal bodies by
mosquito larvae, a protoxin is released. This is broken down into an active toxin by the
midgut enzymes of the larvae, which generally die 24 hours after infection.

Bs is active against some species of the Culicidae. Bs shows good persistence in the
organically polluted water habitats typical of Culex species. It shows good efficacy
against some Anopholes species too. It is less effective against Aedes species.

The major activity of highly toxic strains is due to the crystal toxin, composed of two
proteins of 51 and 42 kDa produced during sporulation

The operational use of Bs in vector control programmes was limited by the late
availability of commercial formulations. Formulations of Bs have been used since 1989
against Culex and Anopheles species in several areas of Asia, Africa and America.

Serratia spp

Bacteria of the genus Serratia (Enterobacteriaceae) are often isolated as insect

pathogens. Some strains can be characterized as invasive, facultative pathogens.
But the most important entomopathogenic species, S. entomophila and S.
proteamaculans, are highly specific and consistently cause chronic amber disease in
the New Zealand grass grub, Costelytra zealandica. Once ingested, these bacteria
colonize the insect’s gut inducing a chronic disease state terminating in death of the
infected insect.
They are available commercially in New Zealand.
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Este documento fue elaborado en base a las siguientes fuentes:

 Bajwa, W.I. and Kogan, M. 2006 (http://www.ippc.orst.edu/dir/microbial/bt/)
 BT (Bacillus thuringiensis) Toxin Resources 2007
(http://www.nal.usda.gov/bic/BTTOX/bttoxin.htm)
 Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J,
Bravo, A. and Dean, D.H. "Bacillus thuringiensis toxin nomenclature" (2007)
http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/
 Weeden, Shelton, Li and Hoffmann eds. Cornell University. 2007.
(http://www.nysaes.cornell.edu/ent/biocontrol/pathogens/bacteria.html)
 (http://www.agrobiologicals.com)

Otras referencias
- Bravo, A. y Cerón, J. 2004. Bacillus thuringiensis en el control biológico.
Colombia, Ed. Buena Semilla, 293 p.
- Caballero, P. y Ferré, J. 2001. Bioinsecticidas: fundamentos y aplicaciones de
Bacillus thuringiensis en el control integrado de plagas. España, Phytoma, 318 p.
- Lecuona, R. (ed.). 1996. Microorganismos patógenos empleados en el control de
insectos plaga. Buenos Aires, Mariano Mas, 338 p.
- Jackson, T.A.; Boucias D.G. and J.O. Thaler. 2001. Pathobiology of Amber
Disease, Caused by Serratia Spp., in the New Zealand Grass Grub, Costelytra
zealandica, J. Invertebr. Pathol. 78, 232-243.
- Kheatan, S. K. (ed) 2001. Microbial pest control. New York. USA. Marcel Dekker
Inc. Ed, 300 p.
- Regis, L.; Silva-Filha, M.H.; Nielsen-LeRoux, C. and J. F. Charles. 2001.
Bacteriological larvicides of dipteran disease vectors. Trends in Parasitology 17
(8): 377-380.