If spores were produced you could try to put them on fresh agar, though I imagine mycorrhizal fungi would be a bit tricky to culture in the first place.

Andrea Dove

you can try to

a. inoculate the spores on fresh PDA media (let the concentration of dextrose be a little high)

b. supplement the PDA with sterilized crude host plant root extract.

Dear,

Andrea Dove

The drying process is critical because if it is completed too quickly, the fungus may lose pathogenicity and virulence or die; nevertheless, if it is completed too slowly, the fungus may become infected by other fungi or bacteria. 10 to 12 pieces of paper filter are placed in a sterile glassine envelope as soon as the fungus has dry. Each envelope is labelled, sealed in a plastic bag, and stored at 4°C or -20°C, depending on the available facilities. A little piece of filter paper is withdrawn from the envelope and placed on fresh medium when a new culture is required.

The novel method is not only dependable, but also economical and simple to implement in any laboratory with limited resources. This approach is used by CIAT to store around 500 insect harmful fungi cultures and 1000 plant pathogenic fungi and bacteria cultures. Purity, pathogenicity, and virulence tests were done on fungus that had been preserved for 5 to 10 years. With a few exceptions, the fungus was easily recovered and had the same pathogenicity and virulence as when it was first preserved.

Andrea Dove, You can try the following medias,

a) PDAY with 5g/L cane sugar + 5ml banana leaf extract (10g leaves extracted)

b) Yeast extract 10g/L + boiled rice water 150ml/L + 7g/L cane sugar (good sporulation usually for Trichoderma spp.)

As Bhavya G. suggested, add the root (sterilised and minced to liquid form) in these mediums for extra support for the fungi, some species may utilize specific components present in the velamen roots

another suggestion is to see if there are any Lichens or mosses associated with the root system of the orchid you are working with, Lichens n mosses produce secondary metabolites which may be a cofactor for the fungi to grow in association with the roots.

Thank you for all the answers. I am working with fungi that do not sporulate (mostly from the family Tulasnellaceae). I can't get my hands on the root extracts although that is a very interesting suggestion which I will keep in mind. I have already tried to put the mycelia onto fresh media with no luck but I will try to increase the carbohydrate source..

Dear, Andrea Dove,

You can take a 0.5 cm disc from the old colony and refresh it by growing it on rich medium such as PDA and incubate it on 25 C for many days. Then you can see the new fungal growth easily.

Dear, Andrea,

As I tell you, you can use medium with high carbohydrate source to promote the fungal growth and obtain a new colony with easy method.

Hope you get a pure colony.

try to recover it on boiled rice or millet

You can revigorate an old culture of non-sporulating mycorrhizal fungus of the Tulasnellaceae family by cutting the agar-based medium into small pieces and macerating them in an agarase formulation (prepared in a buffer solution) with gentle shaking at 25 Celsius degrees for 24-48 hours or until the agar matrix is completely dissolved to free the presumptive surviving mycelium.

Inoculate a series of 100-milliliter flasks containing potato dextrose broth (supplemented with strigolactones and flavonoids; see reference below) with 5-milliliter aliquots of the dissolved agar-based medium containing the surviving mycelium.

Incubate the flasks for 24-48 hours at 25 Celsius degrees with moderate shaking for facilitating the growth of the surviving mycelium.

Take up 2-milliliter samples every 6 hours and measure the absorbance at 600 nanometers’ wavelength with a spectrophotometer to monitor the growth of mycelium.

An increase in absorbance at 600 nanometers’ wavelength confirms the growth of mycelium.

Reference

Orchids and their mycorrhizal fungi: an insufficiently explored relationship | SpringerLink

Orchids and their mycorrhizal fungi: an insufficiently explored relationship

In: Mycorrhiza volume 30, pages5–22 (2020)

According to this reference, the addition of strigolactones and flavonoids in the liquid growth medium may trick the mycorrhizal fungus into believing it is growing in the vicinity of an orchid root and therefore trigger a close symbiotic association with the «neighboring root».

Although this strategy is purely theoretical, it is worth an experimental verification.

Best wishes for a full success.

i had once a success on boiled banana pulpa, from a quite green banana

Thank you for all your very helpful answers. There are some interesting approaches which I will make sure to try.

Já tentou fazer microcultivo?

Agar saubourand entre lâmina e lamínula em câmara úmida, inocular nos quatro cantos do ágar porções da colônia fúngica de interesse.