METHOD OF PREPARATION OF THE LIQUID CULTURE OF TRICHODERMA

This presentation explains the step-by-step method for preparing the liquid culture of Trichoderma, a beneficial fungus widely used as a bio-control agent and plant growth promoter. It covers the selection of mother culture, media preparation (such as molasses and jaggery broth), sterilization, inoculation process, incubation conditions, and methods for mass multiplication. The presentation also highlights the advantages of using liquid culture over solid formulations—such as faster multiplication, longer shelf life, and ease of application in agriculture. Ideal for agriculture students, researchers, and entrepreneurs involved in biofertilizer or biocontrol production.

1. FORMULATION OF BIOPESTICIDES ELP-448 (Group – H) METHOD OF PREPARATION OF THE LIQUID CULTURE OF TRICHODERMA Bappa Chowdhury (2141901294)

2. TOPICS TO BE DISCUSSED Trichoderma and its uses. Different methods: Sugarcane Molasses Method. Rice Bran Broth Method. Bioreactor Method. Shake Flask Method. Hydroponic Nutrient Solution Method. Compost Tea Method. Conclusion.

3. TRICHODERMA AND ITS USES Inhibits the growth of plant pathogens such as Fusarium, Rhizoctonia , and Pythium through the production of antibiotics and enzymes. Boosts microbial diversity and organic matter decomposition in the soil, leading to improved soil fertility. Provides an affordable alternative to chemical pesticides, reducing environmental impact and enhancing sustainable agricultural practices. Trichoderma spp. is a entomo-pathogenic fungus, protects plants from different soil borne pathogen, it effectively manages diseases like damping-off, root rot, and wilt when used as part of an integrated pest management strategy.

4. SUGARCANE MOLASSES METHOD Mix sugarcane molasses with yeast extract in distilled water to create a nutrient-dense solution. Pour the mixture into flasks or bottles. Autoclave at 121°C for 20 minutes After cooling, inoculate with Trichoderma spores under sterile conditions. Incubate at 25–28°C for 7 days with occasional gentle shaking Once active growth is observed, the liquid culture is ready for use or storage

5. RICE BRAN BROTH METHOD Soak rice bran in distilled water and boil to extract nutrients Filter the extract to remove solid particles Pour the rice bran broth into flasks or bottles Autoclave at 121°C for 20 minutes Once cooled, inoculate with Trichoderma spores. Incubate at 25–28°C for 5–7 days under static or mild shaking conditions Collect the liquid culture, ensuring it remains free of large particulate matter

6. BIOREACTOR METHOD Load PDB or MYB into a stirred-tank bioreactor Adjust pH to 5.5–6.5 and sterilize by in-situ autoclaving or filtration. After cooling, inoculate with Trichoderma spores under sterile conditions Maintain temperature at 25–28°C and continuous stirring at 150–300 rpm Monitor and adjust pH and dissolved oxygen levels as necessary Allow the culture to grow for 5–7 days Collect the liquid culture directly from the bioreactor into sterile bottles

7. Shake Flask Method

8. Prepare a hydroponic nutrient solution by dissolving commercially available hydroponic fertilizers in distilled water. Sterilize the nutrient solution by autoclaving at 121°C for 15 minutes or by filtration if heat-sensitive. Cool the solution and inoculate with Trichoderma spores Incubate at 25–28°C for 5–7 days with gentle stirring Use the resulting liquid culture directly for seed treatments or soil drenching HYDROPONIC NUTRIENT SOLUTION METHOD

9. COMPOST TEA METHOD Steep mature compost in distilled water for 24 hours to extract soluble nutrients. Filter the compost tea to remove large organic particles Optionally sterilize by autoclaving at 121°C for 15 minutes Once cooled, inoculate with Trichoderma spores under aseptic conditions. Incubate at 25–28°C for 5–7 days with occasional stirring Collect the liquid culture for immediate use or storage in sterile bottles.

10. CONCLUSION The preparation of Trichoderma liquid culture offers several advantages over traditional solid-state methods, making it a preferred choice for large-scale applications. Liquid culture ensures rapid growth and high biomass production, leading to a higher concentration of viable spores. It provides a controlled environment where factors such as pH, temperature, and aeration can be optimized for maximum efficiency. Additionally, liquid cultures facilitate easy handling, storage, and transportation, making them more suitable for large-scale industrial and agricultural applications. The uniformity of the culture enhances the effectiveness of Trichoderma as a biocontrol agent and plant growth promoter. With continuous advancements in fermentation technology, liquid culture methods are becoming more efficient and cost-effective, further promoting sustainable agricultural practices.