MICROBIAL INSECTISIDES

Why these???

1. Bacillus thuringiensis

 Commonly known as Bt
A highly specific insecticidal bacterium
B. thuringiensis subsp. kurstaki & B.
thuringiensis subsp. aizawai
Used against caterpillars of the Lepidoptera as
butterflies & moths

B. thuringiensis subsp. israelensis

Used against Diptera as Simuliid blackfly (Vectors
for river blindness in Africa), Fungus gnat larvae &
some mosquitoes (as Aedes spp.)

B. thuringiensis subsp. san diego or B.t. var

tenebrionis
Used against some Coleoptera as Colorado potato
beetle.

 Commercially, available as powders

containing a mixture of dried spores &
toxin crystals
Applied to leaves, etc where insect larvae
feed.
Genetic engineering of the toxin genes
into several crop plants (via
Agrobacterium).

Target pests and crops

American Bollworm (Hellicoverpa
armigera)
Pink bollworm (Pectinophera species)
Spotted bollworm (Erias insulana)
Diamond back moth (Plutela xylostella)
Colorado potato beetle (Leptinotarsa
decemlineota
Vegetables, fruit, maize, small grain
cereals and forests, orchards .

Mode of action
Vegetative cells have endospores and crystals
of an insecticidal protein toxin.
The crystals are aggregates of a large 130-140
kDa protein: A protoxin to be activated
Under normal conditions, highly insoluble So,
safe to humans, higher animals & other insects.
Solubilised in reducing conditions when pH >
9.5: The condition in the mid-gut of lepidopteran
larvae.

 Protoxin is cleaved by a gut protease to produce

an active 60 kDa toxin: Delta-endotoxin.
Binds to the midgut epithelial cells
Creates pores in cell membranes & leads to
equilibration of ions
Gut is rapidly immobilised & the epithelial cells
lyse
Larvae stop feeding
Gut pH is lowered by equilibration with the blood
pH.
Lower pH enables the bacterial spores to
germinate
The bacteria invade the host, causing a lethal
septicaemia.

 Delta-endotoxin has three

domains
Domain I: A bundle of 7 -helices
- Insert into the gut cell
membrane, creating a pore
through which ions pass freely.
Domain II: Has 3 antiparallel sheets - Binds to receptors in the
gut.
Domain III: A tightly packed sandwich - Protects the Cterminus end of the active toxin,
preventing further cleavage by gut
proteases.

Bt toxins and their classification

Bt produces 2 types of toxin
Cry (crystal) toxins, encoded by cry genes (> 50
genes !!!!)
Cyt (cytolytic) toxins, to augment Cry toxins

Strain development
Cry toxins are encoded by genes on 5-6 different
plasmids of Bt
A sea of combinations & Cry toxins why?
Plasmids can be exchanged between Bt strains by a
conjugation-like process
Bt contains transposons (transposable genetic
elements that flank genes and that can be excised
from one part of the genome and inserted elsewhere)

So, commercially, genetically engineered strains

with novel toxin combinations

Plants genetically engineered with Bt gene

Genetically engineering to contain the deltaendotoxin gene from Bt
Bt corn
Bt potato
Bt cotton
Bt soybean
The "downside
Perpetual exposure of insects to toxins
Creates a very strong selection pressure for the
development of resistance to the toxins.

 Advantages in expressing Bt toxins in

transgenic Bt crops:
Level of toxin expression can be very high
thus delivering sufficient dosage to the pest
Toxin expression is contained within the plant
system and hence only those insects that
feed on the crop perish
Toxin expression can be modulated by using
tissue-specific promoters
Replaces the use of synthetic pesticides in
the environment.

In an industrial scale
Produced in controlled fermentor in deep
tanks of sterilized nutrient liquid medium
Endotoxins & living spores are harvested
as water dispersible liquid concentrates for
subsequent formulation.

2. Bacillus sphaericus

 Gram-positive bacterium
Used primarily as a larvicide
An obligate aerobe bacterium used as a
larvicide for mosquito control
Forms spherical endospores
Can be isolated from soil, leaf surfaces and
aquatic systems
Produces a 100 kDa protein that acts as a
larvicidal toxin.
Highly effective against the larva of the Wyeomyia
mosquitoes, drastically reducing their population.

 Effective against Culex spp.

Larvicides are more effective and less
toxic than adult mosquito sprays
Unlikely to result in human exposure

Mode of action
B. sphaericus spores are eaten by
mosquito larvae
Toxins released into the mosquito's gut
Larvae stop eating
Effective against actively feeding larvae,
and does not affect mosquito pupae or
adults.

3. Bacillus popilliae

 Gram-negative spore-forming rod.

Spores of Bacillus popilliae infect larvae of
Japanese beetles (Popillia japonica)

 Spores, residing in the soil are ingested by

beetle larvae
Day 2: Germinate in larval gut.
Day 3-5: Vegetative cells proliferate, attaining
maximum numbers.
Day 5-10: Some penetrate the gut wall and
grows in the hemolymph
Day 14-21: A few spores form larva develops
the typical milky appearance.
Host dies

 Spores are ingested by Japanese beetle larvae

(grubs)
Spores become active bacteria and multiply in
the grubs, which continue to live.
Prevents larval maturation.
When the larvaes bacterial population reaches a
high enough density, bacterial spores are
released to the soil to await ingestion by future
beetle larvae.
Infected beetle larvae die when the spores are
released.
Thus, they greatly decrease the numbers of
grubs and adult beetles, thereby reducing plant
damage.

 Spores also infect larvae of some closely

related beetles

 Advantages
Very narrow host range (they are effective
against Japanese beetles, only)
Complete safety for man and other
vertebrates
Compatibility with other control agents
including chemical insecticides

Disadvantages
High cost of production in vivo,
Slow rate of action,
Lack of effect on adult Japanese beetles
Need for large areas to be treated for effect.