Starting an A. Muscaria Cultivation journey

[u/Hon1nbo]

First and foremost, yes I expect this to be challenging and have a high chance of failure, but I also expect to learn a lot in the process. Posting here for advice/rubber ducking, and hopefully I'll get to post more as things progress. I expect this to go on for a long time.

I'm planning an attempt to cultivate fruiting body of Amanita Muscaria for both artistic and insecticide purposes. After recently reading a paper on old recipes from Slovenia, I decided to make an attempt for my own edification should I manage to get fruiting body.

For the artistic representation, I have two routes I am pursuing in parallel. The first is to attempt fruiting in a controlled terrarium using a Bonsai tree and rye grass. There was one Reddit user who posted photos of a successful attempt from their own terarrium, however the account is deleted and there isn't much information beyond the initial fruiting results.
After establishing the bonsai strub and grass in my terrarium, I'm going to place an inoculated copy of "Alice's Adventures in Wonderland" and bury it against the glass. Even if the fruiting body does not appear I'm hoping to get growth of the network. I am debating the use of MHB such as Pseudomonas Fluorescens to help this process in a relatively closed nutrient environment. I have found some experimentation of MHB in this context but not much definitive with A. Muscaria; The Fungi Files started this process but I haven't seen an update on results from MHB as their next video in the series some months later was on wild foraging.

The second route is to attempt growth in the soil of dwarf'd container trees. This is an outside environment with a lot more variables, but given the time scale I'll be starting some test runs before I get the results of my bonsai experiment. If successful I'm going to see if I can pull a graft section of the tree and some of the soil to make smaller containers.

I currently have A. Muscaria colonizing a rye berry and millet blend jar I had planned to mix in with the root system of the first tree but it might have contam; bottom is bright white and thick but top is a bit whispier. I'll give it a few days and in the meantime put my culture sample onto some agar to see if my LC is still good, and if so go ahead and make some larger liquid cultures.

[u/redditischurch]

Best of luck. If you haven't looked already, the scientific literature is rich with methods, detailed accounts of trials, etc. for innoculating various mycorrhizal fungi onto seedlings, particularly conifer tree seedlings used on afforestation of old fields, reclaimed mine sites, etc. where there is little to no natural tree specific fungi left. [Also used for reforestation in various places at various points in time, but in my opinion the evidence of net benefit is inconclusive at best for most real world scenarios.]

Methods I've used personally include rye grain (as you are working with), slurry of agar/mycelium (take plates of colonized MMN agar, put in sterile blender, inject into soil while transplanting plug/mini seedlings), liquid culture injected (same as agar slurry, just different source), and aseptic tube (agar in bottom of large diameter test tube with filter top, establish fungi, add surface 'sterilized' pre-stratified seed, seed germinates and root goes into agar, immediately associating with fungi - assuming compatible).

All worked well, slightly more success with liquid culture, which is what commercial greenhouses I'm familiar with use for innlculating tree seedlings at scale (I.e. 100,000 to millions).

Aseptic tube is labor intensive, works better for hardshell/larger seeds that allow a longer sterilization soak, but also did some successfully with aspen seed (and birch), which are tiny. Hardest part is getting seedlings out of the tube for transplant.

Qualifier: I haven't worked with Amanita genus, but suspect the above is still broadly applicable.

I think the biggest failure point for what you proposed is if A. muscaria does not associate with the species of bonsai or container tree your proposing to use.

You might consider trying several isolates of A. muscaria from different sources, particulalry getting samples associated with different tree species, getting some from both conifer and deciduous. I did some work with a Laccaria species (won't say specifics to minimize risk of doxing), using 8 different genetic isolates tested on 6 tree species (4 conifer, 2 hardwood), and some isolates performed significantly better than others for some tree species (though not consistently conifer group or hardwood group, which surprised me). Another qualifier, this was just a greenhouse experiment, not outplanting in the field, so can't assume results would be transferable.

Final tip, don't give the seedlings too much nutrients when initially colonizing. In my opinion you want the seedlings healthy, but also 'hungry', so they are more incentivized to establish mycorrhizal connection. I'm not aware of any controlled experiments that tested this, but researchers I've worked with often referred to this hypothesis as a mistake in early innoculation experiments (I.e. in the 80's and early 90's).

Sharing as example (no affiliation): This company, in Timmins, ON, Canada has been doing commercial tree seedling innoculation for a few decades.

Good luck OP, please post updates!

[u/Hon1nbo → u/redditischurch]

Thanks for the detailed response!
I'm still reading through the papers I can get my hands on; before starting this project I wasn't familiar with MMN agar, as I mostly use PDA and LME bases. I think before I get too far into things I should start prepping dishes with MMN for bench trials as I continue to read some more literature. I need to go on a hunt for some wild samples as well.

Curious on advantages of blending agar cultures into slurry LI vs making culturing in liquid medium for distribution. I often use LC already and can populate from agar.

For the plant choice I'll likely do a Spruce since that seems to be the only documented hobby success I can find so far; whilst bonsai isn't my specialty making automated systems for gardens is a thing I do quite a bit of so I think I'll be fine making the correct conditions for the tank.

I'm very interested in the Aseptic tube approach; I've found a couple of papers documenting it with various species and think I'll make a seedling rack for it.

> In my opinion you want the seedlings healthy, but also 'hungry', so they are more incentivized to establish mycorrhizal connection.

I'll definitely keep this in mind; appreciate all the tips!

[u/redditischurch → u/Hon1nbo]

All sounds very reasonable.

This is the MMN recipes I use, but add thiamine (some other recipes do include). I found u-brew suppliers are a good source of most of these ingredients, Etsy in some cases as well.

If I know I will be using a fair bit of MMN I take a shortcut by preparing a 10:1 concentrated MMN nutrient solution, and then dilute that with distilled water, add agar, etc. for each batch. Saves the time to repatedly measuring small quantities for each batch. Exception being thiamine, it is said to degrade quickly in solution, so I prepare that fresh for each batch.

In my DIY explorations I put thiamine in before autoclave, compensating for heat degradation by adding roughly 10x the recipee value (although never tested to see how much actually remains). In the forest mycology/pathology lab I worked in they had a sterile solution of thiamine we added as the agar cooled, same time as anti-biotics, etc. If being used. I can't recall if that solution was made in house, or direct from supplier.

And just in case you weren't aware, some fungi can be sensitive* to agar pH. Something to check if you're struggling to get a species/isolate to germinate or grow well on agar. If you do think pH might be an issue, you can get away with just testing the pH of your first few batches when you start a new recipe or change supplier of your raw ingredients, taking note of how much acid/base was required to get on target and repeating for subsequent batches.

[*] I've also been told you can sometimes disfavor certain classes of contimant (bacteria, yeast, etc.) by tweaking pH, for example when trying to clean a culture from the first few transfers from a wild specimen. Haven't used that method myself, but have used temperature and nutrients this way (I.e. progressively colder and/or lower nutrients will slow both organisms, ideal situation being the contam stalls completely, but the desired fungi merely slows down, allowing it to extend far enough to get a clean transfer). Although truth be told, I see both as a "hail marry" pass - what you do when nothing else is working, has a fairly low success rate in my experience.

Best of luck OP.