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Biocontrol biopesticide
- 1. Y5C10 ENTREPRENEURSHIP IN MICROBIOLOGY Unit – IV Preparation of Biocontrol agent (Biopesticide) Dr. S. Sivasankara Narayani Assistant Professor Department of Microbiology Ayya Nadar Janaki Ammal College Sivakasi, TamilNadu, India 01-09-2020Dr.SS
- 2. Biopesticides • Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides 01-09-2020Dr.SS
- 3. Classes • Biochemical pesticides • Microbial pesticides • Plant-Incorporated-Protectants 01-09-2020Dr.SS
- 4. Preparation • Culture isolation • Purification • Mass cultivation • Preparation of Talc based formulation • Packaging 01-09-2020Dr.SS
- 5. Culture isolation • Maize crop – pull out – root – soil sample • Nutrient agar media • Trichoderma selective media • King’s B Medium • Actinomycetes isolation agar 01-09-2020Dr.SS
- 6. Pure culture technique – Pour plate technique • Dilution preparation upto 10-7 • Serial dilution • 10-3 for the isolation of fungi Trichoderma – Pour plate technique – add pinch of streptomycin sulfate in media before pouring onto petri plate. • Three replicates 01-09-2020Dr.SS
- 7. Cont.. • 10 -6 dilution for isolation of Bacillus – transfer 1ml of suspension to a sterile petriplate – pour Nutrient agar media - solidfy • 10-6 dilution for isolation of Pseudomonas - transfer 1ml of suspension to a sterile petriplate – pour kings’ B media – solidfy • Wrap the petriplates and incubate at RT 01-09-2020Dr.SS
- 8. Cont… • 7 days – fungi • 2-3 days – bacteria • 7 – 14 days – actinomycetes 01-09-2020Dr.SS
- 9. Cont… • Bacteria observed after incubation • Trichoderma – white cottony growth • Subcultured the colonies using PDA for culture maintenance and further studies 01-09-2020Dr.SS
- 10. Cont… • Pick single colony (bacteria) from plate – using buds - simple streak on media ( Three replicates) • Sterilize the inoculation needle – take Trichoderma colony using loop – place on PDA center spot. • Pure colonies - Bacillus, Pseudomonas • Pseudomonas - fluoresce • Bacillus -straighted margins 01-09-2020Dr.SS
- 11. Cont… • Pure colonies of Trichoderma observed after – 3- 4 days • After 6- 7 days - full growth • Actinomycetes after 2 days growth starts • 7 days – clear growth 01-09-2020Dr.SS
- 12. Antagonistic activity • Dual plate technique • Make 8mm mycelial disk by using sterile cork borer • Place mycelial disk Pythium on one side of petriplate – 1cm away from side margins • Make 8mm mycelial disk of Macrophomina • Fusarium • Take biocontrol agent –using buds – streak biocontrol agent – opposite side of the petriplate at the opposite side – perpendicular to the pathogen 01-09-2020Dr.SS
- 13. Cont.. • Bacillus inhibit Pythium • Pseudomonas – Fusarium • Trichoderma – Fusarium • Bacillus, Pseudomonas, Actinomycetes- Macrophomina 01-09-2020Dr.SS
- 14. Mass Multiplication • Nutrient agar broth • King’s broth • Sterilize loop – Bacillus culture – inoculate into nutrient broth – 2 – three times • Pseudomonas culture – inoculate into King’s B broth • Incubate broth in a rotary shaker at 150 rpm for 72 hours at RT • After 72 hours – 9 x 108 CFU/ml 01-09-2020Dr.SS
- 15. Talc based formulation • 1 Kg talc powder – add 10g Carboxy methyl cellulose – 400 ml of bacterial suspension – mix well • Shade dry the product – reduce moisture content - < 20% 01-09-2020Dr.SS
- 16. Molasses yeast broth • Trichoderma – mycelial mat of Trichoderma observed after inoculation • Homogenised mycelial mat of Trichoderma – talc powder – mix – refined product • Commercial product 01-09-2020Dr.SS
- 17. Assessing shelf life of Bioformulation • Serial dilution method • 1g of Trichoderma talc powder – serial dilution upto 10-7 • take 1ml of 10-3 dilution – add plate – pour Trichoderma selective medium • Test Pseudomonas - Kings’B medium and Bacillus - Nutient agar Talc Powder • Colonies were observed 01-09-2020Dr.SS
- 18. Test • Treat with plants 01-09-2020Dr.SS
- 19. Questions to think • Biopesticide • Mechanism behind biopesticide? • Name the ingredients of media which is used for Biopesticide production? • Applications of Biopesticide • What are types of organisms comes under biopesticides? 01-09-2020Dr.SS
- 21. 01-09-2020Dr.SS