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CreonAntigone
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I want to cultivate the ergot in ololiuqui (morning glory) 1
#27486198 - 09/29/21 10:05 AM (2 years, 7 months ago) |
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It was recently discovered that the alkaloids found in morning glory seeds are actually produced by a fungus that lives in symbiosis with the plant. The fungal genus has been named Periglandula, and it is in the ergot family (Clavicipitaceae). This article talks about in-vitro growth experiments, so culturing this fungus should be possible.
I want to culture it because many of the alkaloids in morning glory seem like promising psychedelics but they are hindered by the difficulty of eating seeds and the nausea-causing glycosides within the seeds. I think a better experience could be created potentially by culturing the fungus alone and getting it to produce alkaloids in-vitro.
How will I culture this fungus? I'll try a few different strategies. The fungus appears to live throughout the whole organism, although research suggests that it is more concentrated in the trichomes of the leaves. Also, it must also be present in the seeds since there are alkaloids there. I will use 4% malt extract agar and malt extract LC. I've seen some weird recipes where different things are added to nutrient broth to stimulate the ergot to produce alkaloids, however for the moment I just want to isolate and study the fungus. Also, most people grow Claviceps Purpurea, this is an entirely different species in a different genus so similar information may not apply.
I was considering the following strategies:
-Seeds or leaves dropped straight to agar. These can be either washed or unwashed. I can also try to drop just single trichomes to agar.
-I could take a bunch of seeds and leave them in a jar of sterile water for a time (not sure how long), then use drops of this water to inoculate agar. This will not produce a pure culture but it could be used to later get a pure culture.
Also I should note about safety: Claviceps and related species are very variable in alkaloid content. Some produce alkaloids that aren't as dangerous and are more psychoactive. Albert Hoffman argues that the water-soluble alkaloids don't tend to produce toxic effects while most of the oil-soluble ones do. Also, he says that the ones in morning glory tend to be the water-soluble ones. The fungus in morning glory does not seem to be particularly inclined to produce toxic alkaloids judging from its long and consistent use as a psychedelic. I have not heard of a death from morning glory seeds, but I'm sure it is possible. I'm sure the water-soluble alkaloids aren't 100% safe, just safe-ER.
I will be determining which seeds to use based on the alkaloid content of the seeds. Probably not all seeds contain the fungus since some are inactive. I could test seeds the simplest way possible (consuming them) and then determine promising candidates based on the experience. However, safety of the seeds does not guarantee that any alkaloids produced in culture will be safe. I'll proceed cautiously. I intend to only consume a boiling-water extract of the nutrient broth, never the broth itself - this has the benefit of potentially only extracting the safer and more psychedelic alkaloids over the more dangerous ones which tend to be oil-soluble and may not be captured well in a boiling water extract. Whether this sp. even produces alkaloids in culture has yet to be determined so it may be that the nutrient broth is inactive.
Lastly, of course it is possible to absorb alkaloids through the skin. I'll always use gloves and handle any cultures carefully. I really don't think it is that dangerous though. I think the dangers can be minimized thoroughly.
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half.fast
but not too slow, either



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Re: I want to cultivate the ergot in ololiuqui (morning glory) [Re: CreonAntigone] 1
#27486293 - 09/29/21 11:47 AM (2 years, 7 months ago) |
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Based on my experience I think I’d take a different approach than you’re talking about, or at least try one additional thing, based on these thoughts:
- Seeds and plant material have all been exposed to the environment for a long time, making them much more likely to be carrying contaminating organisms you’d like to avoid.
- The fungus must infect the seeds, which are only produced when the plant matures, so the fungus must grow as the plant grows.
- Since the fungus lives within the plant symbiotically rather than pathogenically it probably has a relatively tiny footprint (i.e. theres a lot of plant material and much smaller amounts of fungus) to avoid consuming the hosting plant. This means that in most parts of the plant the fungus is more or less dormant.
Based on this stuff, this is what I’d try:
- Sprout the seeds between sheets of paper towel soaked in sterile water, enclosed in gallon-sized zip-loc type bags to help keep things “clean”.
- From the relatively clean sprouts you can harvest different bits of plant material to transfer to agar. The parts I would most want would be active meristematic tissue since this seems a likely place to find fungus that is actively growing as well.
That’s about it. Good luck with this project, it sounds pretty interesting.
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SingularFusion


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Re: I want to cultivate the ergot in ololiuqui (morning glory) [Re: half.fast]
#27486333 - 09/29/21 12:33 PM (2 years, 7 months ago) |
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Sounds novel
The discussion of culturing the organism itself is interesting, but whether or not it can be convinced to produce ergotamine alkaloids in laboratory culture setting is a really important point
From my understanding, ergot itself does not, except in the rarest of cases, as Hofmann and his lab found on many separate occasions allegedly while at Sandoz
They eventually procured a strain that would, but they obviously have some resources above and beyond you or I
Please let us know more if you discover some things
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CreonAntigone
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Re: I want to cultivate the ergot in ololiuqui (morning glory) [Re: half.fast]
#27487515 - 09/30/21 01:23 PM (2 years, 7 months ago) |
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Quote:
Nef said:
From my understanding, ergot itself does not, except in the rarest of cases, as Hofmann and his lab found on many separate occasions allegedly while at Sandoz
They eventually procured a strain that would, but they obviously have some resources above and beyond you or I
So I learned more about this, apparently Sandoz was actually growing ergot itself in fields before Hoffman even got there. They isolated strains from their own plants. To quote an article: "Working together with the machinery engineers Bucher Guyer, they designed a multi-needle gun to inoculate rye heads with ergot spores, and a special tractor to harvest the heads. The project was conducted with government approval under wartime protocols of secrecy."
So they started with plants. Maybe I need to grow a lot of morning glory first to see if plants reliable transmit the same alkaloidal profile to offspring.
Quote:
half.fast said: [LIST]Sprout the seeds between sheets of paper towel soaked in sterile water, enclosed in gallon-sized zip-loc type bags to help keep things “clean”. From the relatively clean sprouts you can harvest different bits of plant material to transfer to agar. The parts I would most want would be active meristematic tissue since this seems a likely place to find fungus that is actively growing as well.
I think this is a good idea, I'll try it among other things. It's funny, I just read an article that suggests a similar strategy:
Quote:
When a T. corymbosa or I. asarifolia plant was grown from a surface-sterilized seed on an artificial medium under axenic conditions, only these fungi were associated with their respective host plants and the plants contained ergoline alkaloids (Steiner et al. 2006).
The problem is similar to the one you pointed out, that the growth happens in the plants. What I've read suggests the fungus delivers the alkaloids to the plant. So the mycelium on agar may not make the alkaloids. I wonder if I could devise a way to keep a plant living in culture similar to what they do here, and somehow take the alkaloids from it. I don't know, just spitballing.
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Lemgrub



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Re: I want to cultivate the ergot in ololiuqui (morning glory) [Re: CreonAntigone]
#27487605 - 09/30/21 02:54 PM (2 years, 7 months ago) |
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I tried morning glory seeds for the first time a month ago, then one or two more times since. Hawaiian baby woodrose seeds have the highest content of LSA, something like 1% dry weight I think. Most other morning glories have less than a tenth of the actives in HBW.
I ground 13 seeds finely, covered them in a Erlenmeyer with about 15ml 50:50 eth/water and allowed them to stir with my magnetic hot plate in the dark for 4 hours. I extracted this solution with an aeropress, then washed the seed cake with a few ml of everclear 2X. 5-6 hour trip, feels kinda like being drunk.
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LogicaL Chaos
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: CreonAntigone]
#27487645 - 09/30/21 03:23 PM (2 years, 7 months ago) |
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This thread was moved from Mushroom Cultivation.
Reason: Lets try this forum.
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CreonAntigone
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: LogicaL Chaos]
#27487651 - 09/30/21 03:30 PM (2 years, 7 months ago) |
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Quote:
Lemgrub said: I tried morning glory seeds for the first time a month ago, then one or two more times since. Hawaiian baby woodrose seeds have the highest content of LSA, something like 1% dry weight I think. Most other morning glories have less than a tenth of the actives in HBW.
I ground 13 seeds finely, covered them in a Erlenmeyer with about 15ml 50:50 eth/water and allowed them to stir with my magnetic hot plate in the dark for 4 hours. I extracted this solution with an aeropress, then washed the seed cake with a few ml of everclear 2X. 5-6 hour trip, feels kinda like being drunk.
Thanks for the extraction advice. I might try something similar, not sure what the best way to consume the seeds are. I'm sticking with morning glory for now instead of baby woodrose because morning glory is a more well-known and traditional psychedelic. LSA may be more present in woodrose but that's not the only active, according to Hoffman there is also lysergic acid hydroxyethylamide and several more related alkaloids.This study found ergine, isoergine, ergometrine, ergometrinine, elymoclavine, penniclavine, and chanoclavine. They add interestingly, "Not all varieties of seed tested contained these alkaloids: one which contained a substantial amount also contained alkaloid in the leaves and stem of the mature plant."
I may branch into woodrose at some point. The fungus of woodrose may even be the same one producing more/less alkaloids in a different plant or it could be in the same genus.
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SingularFusion


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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: CreonAntigone]
#27488485 - 10/01/21 09:29 AM (2 years, 7 months ago) |
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absolutely, they started with infecting fields of rye, and this produces input material just fine
but they struggled to isolate a strain that would produce lysergamides without the association with a host rye. Basically without nature happening around it, alkaloid production was nil. They couldn't grow it in a lab directly to harvest alkaloids
as I said, they apparently did eventually find one that would do it. I dunno how much work had to be done, I guess they looked at a lot of things before they found genetics that were suitable
I assume you want the specific alkaloid profile found in morning glory? Otherwise if bulk lysergamides are the end game, surely ergot and rye is a fairly productive method of obtaining them
I look forward to hearing more as you learn more about it anyway man
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Speeker

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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: SingularFusion]
#27489658 - 10/02/21 08:23 AM (2 years, 7 months ago) |
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Not so long ago here I suggested putting some slurry or porridge inside those young leaves, as I had read that the fungus is there in sterile conditions. (It's said to grow on plain old PDA - slowly, but it soon stops altogether. So it needs something from its host.) Now I have read some more and it seems that getting it to grow isn't enough as it gives those alkaloids to the host, maybe it gets some nutrients it needs in return. So you need both, fungus and the host until you find out a perfect substrate for it.
The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) - Leistner 2018, Mycota 15 Ergolinalkaloidvorkommen bei Convolvulaceen - Hellwig 2007 (has several agar recipes mentioned - that didn't work in the long run, I assume.. it's in German. Has some pics etc.. Somewhere I have seen a picture of a I. or T. plantlet growing inside a bottle on some medium and having P. mycelium hanging from it, but it's not in this one. Anybody seen it? Where?) https://bonndoc.ulb.uni-bonn.de/xmlui/handle/20.500.11811/3089
Edited by Speeker (10/02/21 07:37 PM)
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Speeker

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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Speeker]
#27490531 - 10/03/21 01:13 AM (2 years, 7 months ago) |
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Quote:
Speeker said: Not so long ago here I suggested putting some slurry or porridge inside those young leaves, as I had read that the fungus is there in sterile conditions.
Started to test this with a woodrose yesterday. Glued the leaf margins together and injected PSA without Agar (plain sugar - sucrose, instead of dextrose.) That is PSB (Potato-sucrose-broth) Before glueing..
 Glued..
After injection..

The amount of PSB I injected was away too much, like a teaspoon, but it has evaporated now (like how?) or the plant has sucked the liquid into itself - don't know. Anyway I probably should have added agar into it. If it dries that easily it might that nothing will grow there... Also used H2O2 to combat germs. It might have some effect too if this fails.. A flow hood would have been nice...
Edited by Speeker (10/06/21 05:26 AM)
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CreonAntigone
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Speeker]
#27492660 - 10/04/21 06:11 PM (2 years, 7 months ago) |
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Thank you Speeker, I look forward to hearing about the results. What is PSA? I'm not familiar. What do you think the long-term use of the treated leaf would be? Would you take a culture from there to agar or would you be using the leaves as an attempt to grow the fungus on its own? Very interesting.
Taking inspiration from that, I am thinking my plan may be to try and take leaves of an established plant (with proven potency of the seeds) and coat in malt extract. See if that can get the fungi to appear more prominently, and then maybe I can take a transfer from aerial mycelia.
Quote:
Speeker said: The amount of PS I injected was away too much, like a teaspoon, but it has evaporated now (like how?) or the plant has sucked the liquid into itself - don't know. Anyway I probably should have added agar into it. If it dries that easily it might that nothing will grow there... Also used H2O2 to combat germs. It might have some effect too if this fails.. A flow hood would have been nice...
Evaporation is a very good sign IMO. It means it's possible the water was used by something. The leaf is theoretically a network/symbiosis between the fungi and the plant. So any water and any additives (such as the PSA) are going to get sucked up by the mycelium. I'd say keep it wet, it may like a misting now and then.
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Alan Rockefeller
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: CreonAntigone] 1
#27494072 - 10/05/21 09:05 PM (2 years, 7 months ago) |
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I would surface sterilize some leaves with dilute H2O2 and/or isopropanol, and then cut them into tiny pieces with sterilized razor blades, changing and/or sterilizing the blades often as to not transfer around contamination. Then put the leaf bits on agar.
When anything grows, sequence it so you know if it's Periglandula or something else.
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Speeker

Registered: 02/11/04
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Alan Rockefeller]
#27494361 - 10/06/21 05:59 AM (2 years, 7 months ago) |
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Quote:
CreonAntigone said: Thank you Speeker, I look forward to hearing about the results. What is PSA? I'm not familiar. What do you think the long-term use of the treated leaf would be? Would you take a culture from there to agar or would you be using the leaves as an attempt to grow the fungus on its own? Very interesting.
Taking inspiration from that, I am thinking my plan may be to try and take leaves of an established plant (with proven potency of the seeds) and coat in malt extract. See if that can get the fungi to appear more prominently, and then maybe I can take a transfer from aerial mycelia.
I put a plastic bag loosely over it to limit evaporation a bit, one end open. Looks about the same..
>PSA w/o a Modified the earlier post. Basically it was just potato sucrose broth. Don't know what to expect. If it grows well does the mycelium still has like no alkaloids? That is, has it given them to the plant? If so, are them still in the leaf. Also, what if the fungus mainly gives the plant some precompounds, from which plant then further processes them? That would explain that the mycelium doesn't have much actives. Leaves have some, the seeds much more.. But how to make this damn thing to flower??? It has been in my living room like 10 years without flowering. Thanks some of you/us guys putting some extra stuff to the envelopes when swapping stuff. Same way I got a Turbina (or is it now in Ipomoea too?). It's almost dead now and never flowered..
This symbiosis is very interesting. And the experiments made. Grafts (one of my favorite subjects) etc.. Killing the fungus and trying to put it back. Failed at some point, should read stuff again if it has changed. Instead they managed to put some roquefort there instead. Is this the same as in blue cheese? Don't remember all. Would Periglandula like some milk?? LOL
Quote:
Alan Rockefeller said: I would surface sterilize some leaves with dilute H2O2 and/or isopropanol, and then cut them into tiny pieces with sterilized razor blades, changing and/or sterilizing the blades often as to not transfer around contamination. Then put the leaf bits on agar.
When anything grows, sequence it so you know if it's Periglandula or something else.
One could do that. It's just that these Periglandula's grow so slowly and stop later without their host.
Funny how in this article at researchgate the Context quotes describe one month old colonies. Then when one views them in full-text the size is a bit different. Micrometres (μm) instead of millimeters (mm). https://www.researchgate.net/figure/Mycelial-structures-of-P-ipomoeae-A-C-E-and-P-turbinae-B-D-F-on-PDA-culture_fig3_51113597
Edited by Speeker (10/06/21 07:56 AM)
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Speeker

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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Alan Rockefeller]
#27494557 - 10/06/21 10:02 AM (2 years, 7 months ago) |
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Quote:
Alan Rockefeller said: When anything grows, sequence it so you know if it's Periglandula or something else.
If some growth is found I can send you some if you want, just tell how to prepare it etc..
Or is there any easily available chemical for alkaloid detection? A bite or two perhaps? Any volunteers?
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PTreeDish


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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Speeker] 2
#27497532 - 10/08/21 03:50 PM (2 years, 7 months ago) |
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I recently made a video showing how to culture and sequence endophytes from grass. You should be able to use the same methodology. Maybe you'll find it helpful for your project.
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Alan Rockefeller
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Speeker] 1
#27498139 - 10/09/21 02:29 AM (2 years, 7 months ago) |
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Quote:
Speeker said:
Quote:
Alan Rockefeller said: When anything grows, sequence it so you know if it's Periglandula or something else.
If some growth is found I can send you some if you want, just tell how to prepare it etc..
Or is there any easily available chemical for alkaloid detection? A bite or two perhaps? Any volunteers?
Ehrlich's reagent would be a good way to see if there's indole alkaloids in your culture.
If you do get something going on agar that looks promising I'd be interested in sequencing it.
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Speeker

Registered: 02/11/04
Posts: 894
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Alan Rockefeller]
#27498280 - 10/09/21 07:42 AM (2 years, 7 months ago) |
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Quote:
Alan Rockefeller said: Ehrlich's reagent would be a good way to see if there's indole alkaloids in your culture.
If you do get something going on agar that looks promising I'd be interested in sequencing it.
Great! Aloha did sequencing for us years ago. Got some new strains/spp. that way I guess. That a bit funny whitish Mushroompeople C. militaris you sent back then was ok. Only one wasn't. (Maybe I have told this before, I don't remember it all anymore.. )
Still about the same. Leaf margins just getting gray. Smelled the bag, a bit fishy smell maybe. Bacteria? I'll let it be maybe a week or two more depending how it looks. If and when this fails, I might try that agar too to see how it grows..
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Seekerqt
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: PTreeDish] 1
#27499200 - 10/10/21 06:29 AM (2 years, 7 months ago) |
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Quote:
PTreeDish said: I recently made a video showing how to culture and sequence endophytes from grass. You should be able to use the same methodology. Maybe you'll find it helpful for your project.
Wow dude thanks alot!
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Speeker

Registered: 02/11/04
Posts: 894
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Re: I want to cultivate the ergot in ololiuqui (morning glory) (moved) [Re: Seekerqt]
#27503836 - 10/14/21 03:58 AM (2 years, 7 months ago) |
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The plant managed to stretch a tiny gab to the gluing, so the sterility of inside was maybe compromised. So I opened it already (11 days passed). No growth what so ever that I could see. The H2O2 did it job alright.. ?? Might give it another try without H2O2 sometime..
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CreonAntigone
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Re: I want to cultivate the ergot in ololiuqui (morning glory) [Re: CreonAntigone]
#27575044 - 12/09/21 05:21 PM (2 years, 5 months ago) |
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I have further thoughts on this....
I now know a bit more about fungal biology. I know that the fungus here, Periglandula, is almost categorically different than mushrooms: it is in the genus Ascomycota, whereas mushrooms are in Basidiomycota. This fundamental difference is of fundamental importance for cultivation.
Well here is my question about Peruglandula: having studied the ergot species, such as Purpurea, I know that early attempts at cultivation identified two kinds of spores, which were very very different. Conidia, were the asexual spores. Conidia were formed many many times throughout the lifecycle, from earlier germination, within sclerotia, and in the 'honeydew' observed to spread between plants: it was conidia that were responsible. Sometimes conidia of various sizes were made.
Ergot sexual spores however are way more specialized: formed only on specialized organs known as stromata, after overwintering of the sclerotia.
Attempts at ergot cultivation found it was extremely easy to get the fungus to grow the conidia...ordinary cultures, such as the dipping of bread in nutrient broth, induced the formation of conidia... but the formation of sexual spores was very hard. We might find this comparable to the concept of 'fruiting' for the Basidiomycetes... it is comparable though perhaps there is still a world of difference.
Useful examples of fruiting Purpurea were posted by me here: beautiful in every way to be honest. Let me post one here for example:

Well now speaking of Periglandula: where does it 'fruit'? That is to say, where does it produce the sexual spores, ascospores, as opposed to the conidia?
Now I will only speak here of I. Tricolor, since that is the material I have available at the moment: I cannot help but be amazed by the utter similarity between Iponemoea Tricolor seeds and that of ergot sclerotia: the difference is uncanny.


So Ipomoea Tricolor seeds are big, black, oddly-shaped and full of ergot alkaloids... they look a lot like purpurea sclerotia...
So surely the seeds are full of conidia, and if you cultured them in malt extract, I'm sure you'd get a culture, and it'd make conidia... but where does the fruitifacation occur?
Looking at pictures of morning glory seeds online I saw quite a very strange one:

What the hell are these strange tentacles! Demented-looking almost, showing the seeds completely parasitized by an ergot-like fungus: Periglandula?
So what if the seeds are like the sclerotia, overwintering structure, and if they put them in the right spot they might grow little fungal growths which would make more sexual spores? The 'TEK' neccesary might be whatever is needed to get those little tentacles out of ergot: perhaps cold, exposure to low temperatures ...
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