Species belonging to Amanita genus are known to be difficult to isolate in pure culture, usually in our lab to maximize the probability to isolate them we usually use a wide range of different culture media. The best results were obtained using Hagem medium

Dear Samuele, thank you for comment. Would you like to describe the Hagem medium composition? Thank you.

dear Vladimir,

this is the Hagem composition for 1 L ddH2O: 4 g malt extract, 1 g yeast extract, 5 g D-Glucose, 0.5 g NH4Cl, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 0.5 mL FeCl3 (1% aqueous solution), 100 μL Biotin (50 μg/mL aqueous solution), 100 μL Thiamine (1 mg/mL aqueous solution), 15 g agar.

You can also try Growth medium PIAM that consists of 20 g malt extract supple-

mented by vitamins (in 20 g: vitamin C 16 mg; vitamin PP

4 mg; riboflavin 0.6 mg; thiamine 0.4 mg; vitamin B6 0.5 mg; vi-

tamin D 0.002 mg), 2 g peptone from

casein acid digest, 20 g glucose, 0.2 g KH2PO4, 16 g agar-agar,

in trace FeCl3, per 1000 ml distilled water.

have a nice day

Samuele

How do you work around the EM needs of A. caesarea? I am aware of a work by Wolfe et al published in PLoS ONE regarding mycelial growth of saprotrophic Amanita and some EM Amanita (primarily those of section Lepidella). This study did not focus on the appearance of basidiocarps, but bringing forth evidence of Amanita which are not EM. You may try to contact Ben Wolfe in regards to your isolation needs.

Wolfe BE, Tulloss RE, Pringle A (2012) The Irreversible Loss of a Decomposition Pathway Marks the Single Origin of an Ectomycorrhizal Symbiosis. PLoS ONE 7(7): e39597. doi:10.1371/journal.pone.0039597

Thank you Samuele! I hope your suggestion will help us.

Thank you, Cristina, for your answer. I'll read this paper.

My aim is only to obtain a pure culture and to grow a vegetative mycelium.

Here is an old paper discussing segregation of an Amanita from Pinus in culture and then reintroduction of the Amanita culture to the symbiotic relationship anew:

Miller, O. K., Jr., D. T. Jenkins and P. Dery. 1986. Mycorrhizal synthesis of Amanita muscaria var. persicina with hard pines. Mycotaxon 26: 165-172.

The target species name is now Amanita persicina.

Details of its morphology and some genetic data is available here:

http://www.amanitaceae.org?Amanita_persicina

Some time ago (probably more than two (or even three) decades ago), Dr. David T. Jenkins (now retired, I understand) attempted to grow an Amanita in culture.  He sterilized pine or oak rootlets and minced them to small fragments and included them in his culture medium.  He used a vegetative culture (probably of A. perisicina because of its aggressive vegetative growth) to inoculate his plates.  In unpublished correspondence with me he described what he thought was initial growth of an Amanita primoridium.  It did not live very long.

This suggests that something is available external to ectomycorrhizae and associated roots that can be used to help start growth of a basidiome in Amanita.  In Jenkins' experiment the dead roots probably provided the substance(s) for a short time, but (since they were dead) did not provide a continual supply.

Another thought.

Amanitas respond to attempts to culture them in highly varied ways.  Amanita volvata is reported to barely grow at all.

On the other hand the muscarioid taxa such as Amanita muscaria, the "American dominant muscarioid" with its several color variants (one current name is Amanita muscaria subsp. flavivolvata, however, the entity should be treated at species rank), and Amanita persicina of the SE U.S. are all very aggressive producers of vegetative mycelium.  I gather that you are having some difficulty with A. caesarea.  You may be running into a difficulty that applies to more of section Caesareae than just one species.  It would be interesting to know about that.  There is an old Master's thesis from Southern Illinois University (U.S.) by a fellow named Tim Began in which, among other things, he reported his success or failure in attempting to culture several amanitas.  If you have trouble finding this document, I may have an old paper copy.  I am going from memory, but I think Tim's research supervisor was the late Dr. Walter Sundberg.  I believe Began went on to medical school and did not continue his work in mycology.

I found the reference for Timothy Began's thesis:

Began, T. J. 1988. The genus Amanita in southern Illinois including cultural characters and a detailed study of volval anatomy in section Lepidella. Master's thesis, Southern Illinois Univ. v + 127 pp.

I have not found the document however.  But I am fairly confident of my memory on the subject of taxa for which culture was attempted.  Despite the title of the thesis, the cultural aspect of Began's work was not limited to Amanita section Lepidella.

Dear Rodham, thank you so much for your comments and assistance. 

Although my main research interests are related with wood-rotting fungi enzymes participating in lignocellulose deconstruction I didn't leave an idea to obtain the Georgian A. caesarea in pure culture and then to try to grow it submerged fermentation. Therefore, any infi is useful to me. However, I am warring that the thesis above is not available to me.

Kind regards,

Vladimir 

You are very welcome.

I will be looking for the thesis today.

Very best,

Rod

Great, thank you!

Found it.

I'm just going to scan the pages related to cultures.

If you want additional parts of the document, please let me know.  The document will be sent later from another computer.

There are nine taxa for which cultures are reported.  They do not cover all the sections of the genus Amanita, and six taxa are in sections with amyloid spores.

Among the three taxa with inamyloid spores that were cultured were two species in Amanita section Caesareae.   Began says they are noteworthy for slow growth in culture (p. 109).  One is Amanita virginiana, a tiny species with two-spored basidia that falls in the near basal group of clades without bright colored pigments.  Began calls this collection "Began 388"; however, I am confident that it is A. virginiana. The second is a North American species which is called "caesarea" by Began, but cannot be the Eurasian taxon with which you are working.

For some reason no species were cultured in sections Vaginatae sensu Zhu L. Yang or Phalloideae.

If you'd like any further notes on systematic placement of the material cultured, I can provide that information.  You cannot trust the use of European names in this document at all.  In 1988, it was not widely appreciated that North American workers were mis-using some European names such as "Amanita caesarea," "A. rubescens" and "A. gemmata."  Molecular work has indicated that the North American taxa are quite distinct.

Very best,

Rod

The partial copy of Began's thesis is attached.

Dear Rod,

I am so grateful to you for your kind help. I'll read the text carefully to find out any useful to me information.                                                                                                    

With kind regards,                                                                                                                           Vladimir

I was pleased to be able to gather the data.  I hope some of it may be of practical help.

Very best,

Rod

I just noticed  a paper posted on researchgate by Naoki Endo relevant to your question.  He has apparently experimented with growing __Amanita caesareoides__ in vitro via inoculation of Pinus.

 Dear Rod, thank you. I know this paper.

Best regards,

Vladimir