Trichoderma can be isolated on Potato Dextrose Agar medium under aseptic conditions.

Through serial dilution method

Amrutha N Bharadwaj Basic medium 0.2 g of MgSO4 · 7H2O, 0.9 g of K2HPO4, 1.0 g of NH4NO3, 0.15 g of KCl, 0.15 g of rose Bengal,

3 g of glucose, and selective antibiotics and fungicides depending on your target species. Look for reference at

Howell, C. R. (1999). Selective isolation from soil and separation in vitro of P and Q strains of Trichoderma virens with differential media. Mycologia, 91(6), 930–934. https://doi.org/10.1080/00275514.1999.12061103

Elad, Y., Chet, I. & Henis, Y. A selective medium for improving quantitative isolation ofTrichoderma spp. from soil. Phytoparasitica 9, 59–67 (1981). https://doi.org/10.1007/BF03158330

Elad, Y., Chet, I. Improved selective media for isolation ofTrichoderma spp. orFusarium spp.. Phytoparasitica 11, 55–58 (1983). https://doi.org/10.1007/BF02980712

Williams J, Clarkson JMMills PR, Cooper RM 2003. A Selective Medium for Quantitative Reisolation of Trichoderma harzianum from Agaricus bisporus Compost. Appl Environ Microbiol 69:. https://doi.org/10.1128/AEM.69.7.4190-4191.2003

Thank you so much

# Materials Needed:

- Soil or phyllosphere (leaf surface) samples

- Potato Dextrose Agar (PDA) or Sabouraud Dextrose Agar (SDA)

- Antibiotics (e.g., streptomycin or tetracycline) to inhibit bacterial growth

- A selective medium like Rose Bengal Agar or Trichoderma Selective Medium (TSM) that contains antifungal agents to suppress non-Trichoderma fungi

- Sterile water

- Petri dishes

- Sterile pipettes

- Incubator (25–28°C)

- Sterile tools (forceps, spatula, etc.)

[Isolation Procedure]

1. Preparation of Selective Media:

- Prepare PDA or SDA plates and add antibiotics (e.g., 0.1 g/L of

streptomycin or 0.1 g/L of tetracycline) to inhibit bacterial growth.

- If available, use Trichoderma Selective Medium (TSM) which contains

rose bengal, chlortetracycline, and pentachloronitrobenzene (PCNB) to

inhibit competing fungi.

2. Sample Collection:

- For soil samples: Collect 5–10 grams of soil from the root zone of plants,

or from areas rich in organic matter.

- For phyllosphere samples: Cut leaves and wash them gently with sterile

water to remove epiphytes.

3. Sample Dilution:

- Soil sample: Mix 1 g of soil in 9 mL of sterile water. Perform serial

dilutions (1:10) by transferring 1 mL from the original mixture into 9 mL of

sterile water in a series of tubes.

- Phyllosphere sample: Take 1 mL of the leaf wash solution and similarly

perform serial dilutions.

4. Plating the Samples:

- Pipette 0.1–0.2 mL of the diluted sample (soil or phyllosphere) onto the

selective agar plates (PDA, SDA, or TSM).

- Spread the inoculum evenly across the surface of the plate using a sterile

spreader or a glass rod.

5. Incubation:

- Incubate the plates at 25–28°C for 5–7 days.

- Check the plates daily for fungal growth. Trichoderma species typically

grow rapidly and form green or white colonies.

6. Colony Identification:

- Trichoderma colonies typically appear green or yellow-green, with a

powdery or cottony texture. Under a microscope, Trichoderma shows

characteristic branching conidiophores with conidia in clusters.

- If using a selective medium, the growth of Trichoderma is usually

prominent as competing fungi are suppressed.

7. Subculturing:

- Once colonies that resemble Trichoderma are observed, transfer a small

piece of the colony onto fresh PDA or selective medium using a sterile

needle or loop.

- Incubate the subculture plates for 5–7 days to allow for further growth

and purification of the strain.

8. Confirmation:

- Morphological identification under a microscope is the most common

method to confirm Trichoderma.

- If further confirmation is needed, molecular techniques such as PCR

(using Trichoderma-specific primers) or sequencing of the Internal

Transcribed Spacer (ITS) region of rDNA can be employed.

- Maintain sterile conditions throughout the process to avoid contamination.

- Using selective media like TSM will help suppress unwanted fungi and

bacteria, giving Trichoderma an advantage.

- Ensure proper incubation temperature (25–28°C) to favor the growth of

Trichoderma.

See the previous discussion:

https://www.researchgate.net/post/How_do_i_isolate_and_identify_Trichoderma_spp_from_soil_samples_what_methods_should_i_use