1. MICROBIAL INSECTISIDES

2. Why these…???

3. 1. Bacillus thuringiensis

4. 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.

5. 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).

6. 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.

8. 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.

9. 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.

10. 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 β- sheets - Binds to receptors in the gut. –Domain III: A tightly packed β- sandwich - Protects the C- terminus end of the active toxin, preventing further cleavage by gut proteases.

11. 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

12. 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

13. 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.

14. 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.

15. 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.

16. 2. Bacillus sphaericus

17. 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.

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

19. 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.

20. 3. Bacillus popilliae

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

22. 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… 

23. 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.

24. Spores also infect larvae of some closely related beetles

25. 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.