How to culture Phytoverm KE for tick control? - briefly
Inoculate a sterile carrier (e.g., wheat bran or rice) with Phytoverm KE spores, incubate at 25‑28 °C and 70‑80 % relative humidity for 7–10 days, then extract the mycelial mass and blend it into an aqueous formulation for application against ticks. Store the prepared product at 4 °C in sealed containers to preserve viability for up to six months.
How to culture Phytoverm KE for tick control? - in detail
Cultivating Phytoverm KE for tick management requires a sterile environment, a suitable growth medium, and controlled incubation conditions.
Begin with preparation of the substrate. Use a liquid medium composed of glucose (20 g L⁻¹), yeast extract (5 g L⁻¹), peptone (10 g L⁻¹), and a buffer such as potassium phosphate (pH 5.5). Dissolve the components in distilled water, filter‑sterilize, then autoclave at 121 °C for 15 minutes. After cooling to 25 °C, transfer the medium to sterile Erlenmeyer flasks (250 mL) under a laminar flow hood.
Inoculation follows. Introduce a pure culture of the Metarhizium anisopliae KE strain, typically a 5 % (v/v) spore suspension containing 1 × 10⁶ conidia mL⁻¹. Mix gently to distribute spores evenly. Seal flasks with breathable caps to maintain aerobic conditions while preventing contamination.
Incubate the inoculated flasks at 25–28 °C with constant agitation at 150 rpm. Monitor growth daily; the mycelial mass should become turbid within 48 hours, and conidiation peaks between days 5 and 7. Harvest conidia by filtering through sterile cheesecloth, then rinse with sterile 0.05 % Tween 80 solution to separate spores from mycelial fragments.
For scale‑up, transfer the harvested conidia into larger fermenters (10–50 L) using the same medium composition. Adjust dissolved oxygen to 30 % saturation and maintain temperature at 27 °C. Aeration rates of 1 vvm and agitation speeds of 200 rpm support optimal sporulation. Harvest and process the broth as described for the flask culture.
Formulate the final product by suspending the conidia in a carrier such as mineral oil or an oil‑based emulsifier at a concentration of 1 × 10⁹ conidia L⁻¹. Add a stabilizer (e.g., glycerol, 2 % v/v) to enhance shelf life. Store the formulation in opaque, airtight containers at 4–10 °C; viable spore counts remain above 80 % for at least 12 months.
Application guidelines: disperse the suspension uniformly over tick‑infested habitats using a calibrated sprayer, delivering 2–3 L ha⁻¹. Apply during cooler parts of the day (early morning or late afternoon) to reduce UV degradation. Re‑treat at 14‑day intervals until tick populations decline below economic thresholds.
Key quality‑control checks include:
- Spore viability: >90 % germination on PDA after 24 h.
- Purity: absence of bacterial or fungal contaminants in culture plates.
- Moisture content: <5 % in the final formulation.
Adhering to these procedures yields a consistent, high‑potency biocontrol agent effective against ticks.