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Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants
    #29620490 -

I saw this fascinating study about a strain of Psilocybe ovoideocystidiata isolated from the leaves of a wild-growing Cannabis plant in India.

And I also wonder, what other psilocybin-producing plants form associations with plants and live inside them? Semilanceata is known for living in grass roots, and seems to confer benefits to the grasses.

Psilocybe-inoculated plants would be an interesting and sustainable way of propagating mushrooms outdoors, as if it was done right the mushrooms may keep coming up as long as the plants live. But what other species might this apply to? As far as I know, the dung and woodlovers don't display endophyte activity.

I also find it quite comical that this mushroom was found on the cannabis plant, and so is it just a coincidence or is there a reason these two drug-producing species formed a symbiosis? Could the cannabis tissue that is colonized by one of these mushrooms contain a small amount of psilocybin?

Full text of the article someone uploaded to scribd. Phylogenetic tree from the article (it seems it is actually pretty closely related to cubensis...)


Edited by CreonAntigone (07/09/26 08:09 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29620617 -

Thanks for posting that! I missed that P ovoideocystidiata paper but thought it wouldn't be long before more Psilocybes were found to be endophytes along with P semilanceata.

Something interesting I came across is that there's another indole producing fungus from the Neotyphodium genus that's an endophyte of a grass species called Achnatherum robustum, that produces LSD like substances that puts horses and cattle that graze on the grass into a stupor that lasts several days. The host grass is called Sleepy Grass because of this effect.

So it would be really interesting to analyse the leaves of the grasses that host P semilanceata colonies and see if there's any psilocybin or psilocin showing up in the grass leaves. Who knows? If a few mouthfuls of Sleepy Grass can fell a horse, you might even be able to get high on the grass rather than wait for the fruits in Autumn.


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Edited by Dave Bennett (07/09/26 12:30 PM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29620872 -

Dave Bennett said:
So it would be really interesting to analyse the leaves of the grasses that host P semilanceata colonies and see if there's any psilocybin or psilocin showing up in the grass leaves. Who knows? If a few mouthfuls of Sleepy Grass can fell a horse, you might even be able to get high on the grass rather than wait for the fruits in Autumn.


I think more likely you'd find it in the roots rather than leaves.

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29621074 -

Oh snap. One more reason I Wana try growing them sounds like a fun experiment with my autoflowers


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An enigma some caerulescens n a mono 😎

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29621133 -

The hypothesis would be that indoles are a grazing deterrent which would be beneficial to Liberty Caps and their host grass species so you'd have to look for the indoles in the grass leaves.

Substances can be translocated from roots to leaves but we still don't know what the mycelium was doing when it invaded cells in grass roots in the Keay and Brown paper that observed that the mycelium behaved like an endophyte when it encountered grass roots.

There are limitations about what can be inferred from the results of that paper too. The grass roots were the only organic material in the experiment so the hyphae could have been just exploring it's environment, trying to digest the grass roots, parasitizing the root cells or be initiating a symbiotic relationship with the grass plants. We don't know if the mycelium invaded the roots and then would have spread into the grass leaves as the roots were the only part of the grass examined in the experiment which I think only ran for a matter of days.

I used to favour the root endophyte hypothesis because the electron microscope pictures of the invaded root cells in the "Colonization by Psilocybe semilanceata of roots of grassland flora" paper look almost identical to electron microscope pictures of root cells being invaded by endophytes that have a mycorrhizal relationship with plants.



The pic on the left is a typical electron microscope picture of a root cell being invaded by the hyphal peg of a mycorrhizal fungi species with a haustorium like structure forming to initiate a zone of nutrient exchange with the root cell.

The pic on the right is a diagram of an invaded root cell showing changes in the cell and the various zones that form around the hyphal peg and haustorium inside the root cell.

And the pic in the middle is an electron microscope picture of P semilanceata hyphae penetrating grass root cells from the Keay and Brown paper mentioned above.

Since then though I've had some correspondence with one of the leading experts in the field of grassland fungi who does a lot of eDNA work on grassland soil samples where Liberty Caps commonly fruit and they told me that they'd never found P semilanceata DNA in their soil samples despite them containing lots of grass roots. They ascribed this to the mycelium being confined to the litter layer on or just under the surface of the soil and thought that this could indeed indicate that P semilanceata is endophytic in the leaves and stems of it's host grasses in nature.

Regardless of where it resides I think there's definitely some sort of beneficial symbiotic relationship going on between Liberty Caps and certain grass species. I noticed in some of my experimental grows that the grass grew much better in the pots that contained the most mycelium.





It would be relatively easy to prove that they are a leaf endophyte by putting a sample of grass stem or leaf from a clump of grass that hosts Liberty Caps on agar and then sequencing any mycelium that grew out of the sample. It's something I intend to do with the grass that's growing in my outdoor Liberty Cap pots and trays at some point in the future.


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Edited by Dave Bennett (07/10/26 03:18 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29621149 -

Fascinating stuff, thank you. Especially that correspondence with the researcher.

To me the middle container looks like it has colonized roots, similar to the ones found in invitro mycorrhization studies. Especially the part lining the bottom. Perhaps you could cut a small section from those roots and try plating that too.

I guess like your post says, we may not know exactly how they associate with grasses yet. But an argument in favor of them being beneficial in the rootspace is that one study found invitro they suppressed the growth of a root rot fungus, Phytophthora cinnamomi. Perhaps the indoles serve as mold killers? Allowing the host plant to be wet all the time and not succumb to bacterial or fungal disease. 

I think the alkaloids that discourage grazing tend to be clavicepticae, but I'm not sure if psilocybin would discourage grazing. I would imagine that unless the amount in leaves is extremely high, most grazing animals would not feel that intensely, or even have a pleasant experience. I've read posts here talking about cows that enjoy eating psilocybin mushrooms.

For insects though it's a different story, and I could see it discouraging insects from eating the grass.

Edited by CreonAntigone (07/10/26 05:23 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29621196 -

When I first got into trying to grow them it was on the back of realising that the Keay and Brown paper had been widely misinterpreted and it didn't show that they fed on decaying grass roots at all as they'd only invaded young healthy roots which showed no signs of senescence or decay. So I concentrated on inoculating living grass roots of the species who's roots they invaded in the Keay and Brown paper with the intention of growing on the grass plants in an acidic pH balanced compost to see if any Liberty Caps fruited from the clumps without the benefit of artificial nutrients like grain. I think the tub I planted up is five or six years old this year. No fruits so far but I haven't given up on the experiment yet as the mycelium is very slow growing and may take years to achieve a colony that's capable of fruiting.



I did stick a small sample of grass stem from the tub on agar a couple of years ago just to see what would happen and white fluffy mycelium did grow out of the grass sample (at 12 O'clock) before it was quickly overtaken by Trich but it does indicate that a fungal endophyte is present in the leaf tissues of the grass.



I know people say that animals purposely ingest Psilocybe mushrooms but I've never seen any credible evidence that indicates that actually happens. In all my years of picking Liberty Caps I've never found any that had their caps grazed off by cattle or sheep just leaving the bottom of the stems. I have noticed that the grass clumps they fruit from don't tend to get grazed by animals though. You can see it here in this pic. The grass clump that's hosting the Liberty Cap colony has remained ungrazed since it started flowering as indicated by the all the dried flower spikes but the surrounding grass outside the ring of fruits is much shorter (which indicates grazing activity) with hardly any flower spikes present at all.



The alkaloids wouldn't have to be present in the grass leaves in amounts that would have a psychoactive effect on animals to have a deterrent effect. Psilocybin, psilocin and psilocin oligomers all have a really bitter taste so the fungus could just be supplying the grass with enough alkaloids to taste bitter enough not to be eaten by grazing animals. What ever it is, something is definitely putting them off eating grasses that host Liberty Cap colonies from my observations.


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Edited by Dave Bennett (07/10/26 07:21 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29621207 -

Are those a Carex sp?

Also, I want to ask why you think most previous attempts to grow liberty caps using grainspawn failed, while yours succeeded. What was done wrong that you corrected? Or did you luck out with a good culture/properly develop the culture?

I feel like I've seen most of the things you describe in your tek tried by others and usually it doesn't result in success.

Edited by CreonAntigone (07/10/26 08:07 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone] * 1
    #29621255 -

I think it's Smooth Meadow grass, Poa pratensis, in the pic which is one of the species the hyphae invaded in the Keay and Brown paper.

I think the main reason my Liberty Cap Tek works so well is nutrient depletion triggering an all out fruiting response. The less grain spawn I used the better they fruited and the greater the number of fruits in the pots so I think other people probably failed in the past because they went too heavy on the grain spawn and bulk sub. The outdoor trays that I spawned lasagne style with a lot of grain spawn took ages to fruit and produced bigger fruits but there were a lot less of them than in the PP5 pots.

When lots of nutrients are available and there's lots of space to expand the mycelium is splitting its resources between growth and colony expansion, storing nutrients to get through the Winter and the decision to fruit. When nutrients and space are restricted it's got no choice but to go for an all out fruiting and use spores to escape its confines and find a new place to grow and fruit.

Going back to Psilocybes that could be endophytes, I think the subaeruginosa complex has to be given strong consideration. I was chatting to someone who found Wavy Caps growing directly from the exposed broken roots on the root plate of a recently fallen Sweet Chestnut tree where the roots had been broken as they pulled out of the ground. They'd also found a tiny Wavy Cap growing from a twig that had fallen off a tree onto tarmac that hadn't been in contact with the soil at all.

If the subaeruginosa complex are plant and/or tree endophytes it would certainly explain how they spread around the world so easily in imported plants or trees and their appearance and success in wood chip beds because the mycelium would have already been present in the living wood of host trees before it was chipped. And if you carefully dig up dune cyans and azzies you find that that a lot of the time they're fruiting directly from the Marram grass rhizomes too which often indicates an endophytic relationship with the host plant. Like in these pics someone sent me.





And going back to Liberty Caps, check this picture out. It's a Liberty Cap fruiting from a dead reed stem that was about 6 to 8 inches above soil level. So P semilanceata mycelium seems to be happy inside dead plant tissues above the surface of the soil.



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Edited by Dave Bennett (07/10/26 10:00 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29621284 -

Growing right from the root seems like a pretty good sign.

I would also suggest the possibility that many coprophilous psilocybe are endophytes, but that this aspect of their lifecycle is not as studied. It's just a possibility, though. But the relatively close relation of Cubensis to Ovoideocystidiata is suggestive.

Quote:
Several coprophilous fungi have also been reported to have the ability to live as plant endophytes (Herrera et al. 2011; Newcombe et al. 2016). Occurrence of coprophilous fungi from surface-sterilised plant tissue had been reported before, however it had remained unclear whether these fungi were incidental contaminants that were not killed by alcohol or bleach (which may even have enhanced spore germination, just like gut passage might achieve) or whether these fungi were true endophytes. Newcombe et al. (2016) provided evidence for an endophytic life style of Sordaria fimicola and also for negative fitness effects on the growth of the grass Bromus tectorum. However, Griffith et al. (2017) reported that the same fungus was more common on the same grass species under more drought-prone environments, suggesting a possible role of the endophyte in drought tolerance. It has also been reported that Sordaria fimicola can reduce symptoms of the cereal disease take-all in rye and wheat (Dewan et al., 1994). The  endophytic life style, which likely causes higher fungal selectivity towards certain plant species, could also be an explanation for the positive correlation between the number of plant species foraged by herbivores and the number of coprophilous species found on the dung of these herbivores. Endophytic occurrence has also been reported for subcoprophilous fungi such as Psilocybe semilanceata (Keay & Brown, 1990).

Kuyper, van Peer, A.F., Baars, J.J.P., 2021. Coprophilous fungi; Closing the loop: improvingcircularity with manure-loving mushrooms/

A possible synergy in that lifecycle: plant matter which is colonized and gets consumed may then turn into viable mycelium once it gets eaten and becomes dung. That is the case for the Cheilymenia. Very distantly related (Ascomycetes) but I put it here to suggest a possible lifecycle.

Edited by CreonAntigone (07/10/26 11:53 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29621350 -

That's a really intriguing possibility that I haven't considered before. I don't know whether you saw this pic I posted on another thread but I found that cube mycelium naturally grows down into the soil where it would of course be in close contact with grass roots. If you zoom into the left side of the picture you can see the mycelium has swathed the bottom of a grass seedling and that bright white thick strand above left of the fan of mycelium is where it's followed the root down into the soil.



I can feel a new project coming on. I'm going to make up a pot of Wavy Cap spawn with fresh wood chips and compost and plant a suitable tree seedling into it and then after a few months try and culture the mycelium from a twig sample above ground so there's no chance of the mycelium being on it's surface as would be the case with root samples.

I think we may be on to something here.


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Edited by Dave Bennett (07/10/26 01:28 PM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29621708 -

Grass looks a bit less healthy in that one, wonder if cubensis is not as kind to the grasses... Or maybe the environment is a bit too humid.

I mixed cubensis spores with water and a drop of honey, to help start the germination process. Then I soaked rye grains in the water for 1 day. I am going to germinate the rye grains, and afterwards test if the blades of grass contain a fungal endophyte on agar. I will also look for some signs of mycorrhization.

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29622057 -

That's a great idea! P semilanceata colonised rye grass plants roots in that paper, although to a lesser extent than the native wild grasses so it should be Psilocybe friendly.

Alder seedling ordered here so the Wavy Cap experiment should begin next week.

Are you intending on getting any mycelium that grows out of the rye leaves sequenced?


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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett] * 1
    #29622398 -



All the bispored species of Copelandia I have grown pins better with grass and beneficial microbes.  Those are the large group at the bottom after the bisporus/cyanescens split (note that a lot of these names are just what they were in genbank, the China tropicalis near axfordii is likely a mis-ID for example).

And I've also noticed they tend to follow the roots down and cord more around them.

But I'm not sure that they actually penetrate the grass cell structure.  Or how to observe that with the equipment that I have.

Tried scoping a root once and it was a bit of a mess.

But even if they're not endophytes I imagine there's mutualistic relationships, where the mycelium protects the grass from parasitic fungi.  And the mycelium benefits from the aeration and microbes that live on the grass roots.  While also finding some protection from the elements for the pins.


--------------------
And if I only could
I'd make a deal with God
And I'd get him to swap our places
I'd be running up that road
Be running up that hill
With no problems

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: tree frog]
    #29623395 -

Noticed fuzz on grass directly in my caerulescens patch visible to the naked eye :0 assuming its mycelium because it resembles it alot and the grass with such fuzz in that patch happen to be taller and newer growth than the grass from elsewhere on the property.

Another thing to note I added 0 extra nutrients to the soil or substrate or spawn just spawned to the ground :rockon:  so no reason for the grass to be bigger. There's more nutrients in soil elsewhere on the property and plenty of water elsewhere too so nutrients and water are not a limited for the grass.

Can only assume there's a symbiotic relationship somehow if u have and ideas on how to verify this I'm all ears 😎


--------------------





An enigma some caerulescens n a mono 😎

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: tree frog] * 1
    #29623426 -

You need to treat the roots with the microscopy stain Lactophenol Cotton Blue to stain any internal hyphae which makes them easy to see under the microscope. There's step by step root prep and staining methods available online and it's pretty simple to do.

This was a grass root from my outdoor Liberty Cap tub I just cleaned and stained a few years ago that I think shows the hyphae (blue squiggles) growing between the root cells under the scope.



It would be interesting to put a sample of the grass leaves from one of your Copelandia grows on agar just to see if any mycelium grew out of it. Again, there are simple methods of surface sterilising the leaves or roots for growing out endophytes available online.


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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: Dave Bennett]
    #29623467 -

I'll give it a try this fall. I don't currently have any of those cultures going.

If it moves into the leaves, it should be possible to get it going on water agar.  Especially if I use a species that has distinct mycelium like chlorocystis.  Just so I can verify without having to grow it back out or sequence it.

Also I appreciate the information Dave ♥️


--------------------
And if I only could
I'd make a deal with God
And I'd get him to swap our places
I'd be running up that road
Be running up that hill
With no problems

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: tree frog]
    #29624013 -



After soaking these rye seeds (secale cereale) in the spore water, I germinated them in a bag with a wet paper towel.
Now they will grow a terrarium for a while to assess their health (salsa jar with the lid slightly unscrewed for ge). Coir was used as the substrate and not soil to reduce the risk other organisms could get in instead and confuse the experiment.

Later I will plate parts of the grass blades and see what grows. I'll also look at how well these plants are doing health-wise.

Despite these seeds having a high germination rate for me normally, I got very few to germ compared to the number of seeds I used. I wouldn't soak it, if I could do it over. I wouldn't start the spores either. I think the mushroom spores which were already started were prone to decompose the seeds too much during the soak. If I were to do it over I'd do no soak and a few spores added to a paper towel used to start the seeds. Nonetheless, I expect these seeds that did get going to be perfectly healthy.

Edited by CreonAntigone (07/14/26 03:30 AM)

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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: CreonAntigone]
    #29625578 -

CreonAntigone said:
I saw this fascinating study about a strain of Psilocybe ovoideocystidiata isolated from the leaves of a wild-growing Cannabis plant in India.

And I also wonder, what other psilocybin-producing plants form associations with plants and live inside them? Semilanceata is known for living in grass roots, and seems to confer benefits to the grasses.

Psilocybe-inoculated plants would be an interesting and sustainable way of propagating mushrooms outdoors, as if it was done right the mushrooms may keep coming up as long as the plants live. But what other species might this apply to? As far as I know, the dung and woodlovers don't display endophyte activity.

I also find it quite comical that this mushroom was found on the cannabis plant, and so is it just a coincidence or is there a reason these two drug-producing species formed a symbiosis? Could the cannabis tissue that is colonized by one of these mushrooms contain a small amount of psilocybin?

Full text of the article someone uploaded to scribd. Phylogenetic tree from the article (it seems it is actually pretty closely related to cubensis...)




After reading that article I find nothing compelling to suggest that they didn't inadvertently introduce the psilocybe species during plating or that it wasn't just spores on the leaves. They didn't elucidate their methods at all. I wouldn't even consider this real until further compelling research is done.


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Re: Psilocybin-producing mushrooms which are endophytes or otherwise symbiotic with plants [Re: bodhisatta]
    #29625586 -

bodhisatta said:
After reading that article I find nothing compelling to suggest that they didn't inadvertently introduce the psilocybe species during plating or that it wasn't just spores on the leaves. They didn't elucidate their methods at all. I wouldn't even consider this real until further compelling research is done.


I agree they don't say much.

There is just one sentence:

"The isolation of the endophytic macrofungi was carried out on modified potato dextrose agar (PDA) by surface sterilization and spread plate method [7]."

It's possible they cited that article to say they follow the method of the other paper exactly. If that's the case, I followed the citation to see what they did in the other paper.

Isolation of endophytic fungi from South African plants, and screening for their antimicrobial and extracellular enzymatic activities and presence of type I polyketide synthases

Quote:
Fresh plant leaves, roots and stems were immediately rinsed under running tap water for 5 min to remove soil. Then plant parts were cut into 0.5 × 0.5 cm2 segments. The following step was the surface sterilization, where different plant segments (roots, stems and leaves) were treated with 70% ethanol for 1 min, followed by a strong surface sterilant (3.5% sodium hypochlorite) for 5 min. The material was washed again with 70% ethanol for 30 s followed by several rinses in sterile distilled water for 1 min to remove residual sterilant (Abdalla and McGaw 2018). The sterilized segments were inoculated onto Potato Dextrose agar (PDA), which was supplemented with penicillin G (30 mg/L) and streptomycin sulfate (30 mg/L). The Petri dishes were incubated at 28  °C for two weeks. Additionally, a control sample taken from the water washings of the surface-sterilized segments was streaked onto PDA (antibiotic-free) and incubated under the same conditions. The primary growth of the fungi was isolated after 14 days and several sub-culturing steps were performed using PDA and incubated at 28  °C for a further 7 days until the fungal strains were purified



Of course we can't assume they used this method exactly, since they provide little info. And an important step is using the water washings as a control group for an endophyte vs what is just found on top of leaves.

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