MICROBIAL INSECTISIDES

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MICROBIAL INSECTISIDES
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Why these???
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1. Bacillus thuringiensis
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• 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.

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• 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).

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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 .

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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 gt 9.5
  The condition in the mid-gut of lepidopteran
  larvae.

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• 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.

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

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Bt toxins and their classification

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

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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

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Plants genetically engineered with Bt gene

• Genetically engineering to contain the
  delta-endotoxin 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.

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• 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.

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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.

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2. Bacillus sphaericus
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• 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. 

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• Effective against Culex spp.
• Larvicides are more effective and less toxic than
  adult mosquito sprays
• Unlikely to result in human exposure

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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.

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3. Bacillus popilliae
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• Gram-negative spore-forming rod.
• Spores of Bacillus popilliae infect larvae of
  Japanese beetles (Popillia japonica)

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• 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?

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• 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.

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• Spores also infect larvae of some closely related
  beetles

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• 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.