Transcript Bt - EngineeringDuniya.com

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 α-helices
- Insert into the gut cell
membrane, creating a pore
through which ions pass freely.
– Domain II: Has 3 antiparallel βsheets - Binds to receptors in the
gut.
– Domain III: A tightly packed βsandwich - 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 larvae’s 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.