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Stipe-n Cap


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Axenic inoculation versus symbiotic bacterial inoculation 3
#26872959 - 08/10/20 09:39 AM (3 years, 5 months ago) |
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Bacillus inoculations have been widely used in the production of Pleurotus ostreatus, Pleurotus eryngii, Agaricus bisporus, shiitake, etc.
Bacillus megaterium has been shown to stimulate primordia formation, (According to Park and Agnihotri (1969)) Bacillus thermofibricolous: if introduced at spawning, inhibits the growth of competitor molds in rice bran/sawdust spawn (according to Steineck (1973)) Bacillus subtilis QST713 has been used as a biofungicide coating on grain spawn to prevent Trichoderma aggressivum: Source provided at the bottom of the post.
Many bacteria have been shown to be beneficial in primordia and fruit body formation, hyphae growth etc. Though I'm sure this practice is out of reach for most hobby growers for obvious reasons, does the pursuit of strictly axenic spawn and substrate production represent a limitation to furthering our understanding of the mushroom life cycle. We may be able to increase productivity, increase the instances of fruiting otherwise stubborn to fruit species, and a potential reduction of common seasonal contamination problems within the community...I think that it's within reach of some of the more experienced growers here that have connections to lab techs.
Growing edible mushrooms: a conversation between bacteria and fungi: "Although fungi have coexisted and interacted with bacteria since the earliest stages in fungal evolution, we still understand relatively little about their interactions. Culture independent techniques have allowed researchers to establish that in a natural context fungi‐selected microbiota exist along the mycelial surface and within proximity to mycelia (Halsey et al., 2016). The environment within and surrounding fungal hyphae, sometimes known as the mycosphere, influences and is strongly influenced by bacterial communities, to the extent that many macrofungi are unable to produce fruiting bodies in a sterile environment (Noble et al., 2003, 2009). Unsurprisingly, bacteria‐fungi interactions have a significant impact on the productivity of commercial mushroom farms."
Source: https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1462-2920.14765

Bacteria sequenced from fermented woodchip substrate colonized with the above species of mycelium. The bacteria present in the substrate formed mutualistic-beneficial partnerships within the mycelial-mass resulting in a beneficial immunological effect for both colonies (bacterial and fungal). Source: Stamets.
I believe we could benefit by incorporating these practices that are somewhat common in the commercial mushroom community. Access and legality obviously representing a limiting factor.
This article is a perfect example of the process used to identify and isolate symbiotic bacteria from any species of mycelia.
https://academic.oup.com/femsle/article/218/2/271/530921
"Bacteria were collected from cultures of P. ostreatus following mushroom production on samples of composted and noncomposted cotton plant waste products. Samples were selected from spent culture medium that had previously produced either high or low yields and the microorganisms were classified into four groups (C-H, C-L, NC-H, and NC-L) according to whether they had been isolated from composted (C) or noncomposted (NC) plant waste, with, high (H) or low (L) yields."
The selected species in this case was P. ostreatus, however it could have easily been Pan. Cyanescens with samples taken from spent composted manure substrate rather than spent composted cotton...
"Other cultures of bacteria were obtained by direct sampling of mycelia produced in composted waste cotton, and were collected after the first harvest of P. ostreatus at 3 to 7 cm depth below the surface of the mushroom culture medium. The mycelia were carefully isolated by hand, using tweezers.."
Ten colonies of fluorescent Pseudomonas were selected and isolated from the mycelial plane of P. ostreatus.
P. ostreatus were grown on TSB agar with each bacteria bacterial To observe the effects of selected fluorescent pseudomonad cultures on growth of axenic cultures of mycelium.
The result:
Inoculation with fluorescent Pseudomonas spp. significantly promoted the development of the mycelium of P. ostreatus, the formation of primordia, and the development of P. ostreatus basidiome.
Hypothesis:
It is likely that the Psilocybe producing Coprophilous/Saprotrophic species have similar relationships with the bacteria in their respective substrates. Isolating those bacteria and Understanding their effect on health and growth will greatly advance our understanding of these species.
The composted substrates used to grow exotic Coprophilous and Saprotrophic species are the best candidate's for investigation into which bacteria form beneficial partnerships with their respective mycelial colonies as shown above.
Most of that process is out of the reach of most growers without lab connections, however if using the sampling scheme above, one could collect samples from within the spent manure/wood substrate and attempt to grow out some colonies on agar: Once colonies form, streak to new plates to be sent away for sequencing. This will at least tell you whats in the substrate. Isolating those bacteria and culturing them separately on agar to observe the interactions between them and mycelium on agar, well, this is an entirely different problem that will need to be solved.
Bacterial samples can be sent here for sequencing:
https://www.genewiz.com/en/Public/Services/Sanger-Sequencing/Direct-Colony-Sequencing?sc_device=Mobile
Some source material for your reading pleasure. There is lot's of this material online, these are just a few. This post constitutes a proof of concept for active species.
The nature of the microbial stimulus affecting sporophore formation in Agaricus bisporus (Lange) Sing
https://www.researchgate.net/publication/229916455_The_nature_of_the_microbial_stimulus_affecting_sporophore_formation_in_Agaricus_bisporus_Lange_Sing
Impact of Bacillus subtilis QST713 mushroom grain spawn treatment on yield and green mould control
https://www.researchgate.net/publication/330711315_Impact_of_Bacillus_subtilis_QST713_mushroom_grain_spawn_treatment_on_yield_and_green_mould_control
Microorganism of Pseudomonas putida which stimulates the growth of mushroom and method for culturing mushrooms using the same
https://patents.google.com/patent/KR20040078027A/en
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maxmush
Always learning...
Registered: 06/13/20
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#26872993 - 08/10/20 10:01 AM (3 years, 5 months ago) |
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tagged for interest
-------------------- Disclaimer: all information presented is intended for educational purposes only. All photos are only representations and not directly from the user.
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Stipe-n Cap


Registered: 08/04/12
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27141941 - 01/11/21 12:28 PM (3 years, 17 days ago) |
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"A biofungicide based on Bacillus subtilis QST713 was tested for impact on yield and efficacy against a Trichoderma aggressivum f. europaeum T77 strain from Serbia by coating mushroom grain spawn and comparing the results with the chemical fungicide prochloraz manganese in a mushroom growing room. The tested B. subtilis QST713 strain did not inhibit mycelial growth of Agaricus bisporus in plots free of the pathogen, showing an impact on yield of 91.95%, which was not significantly different from an untreated control. As for the efficacy of the fungicides used against T. aggressivum f. europaeum T77, there were no significant differences between a prochloraz manganese casing treatment, and B. subtilis QST713 coating on mushroom grain spawn, as the efficacy was 70.37 and 53.09%, respectively. These results implied that the biofungicide based on B. subtilis could serve as a harmless alternative to synthetic fungicides in mushroom production, especially during serious compost green muold outbreaks caused by T. aggressivum. Furthermore, the biofungicide should be applied alone because an antagonistic reaction was detected between the fungicide prochloraz and B. subtilis QST713."
https://www.researchgate.net/publication/330711315_Impact_of_Bacillus_subtilis_QST713_mushroom_grain_spawn_treatment_on_yield_and_green_mould_control
I will be sourcing B. subtilis QST713 from Serenade antifungal spray to begin my trials. My intention is to inoculate petri dishes with B. subtilis QST713 using an inoculation loop and then transferring bacteria colonies to LC; LC will then be used to coat grain spawn.
   
First round of testing will be to determine if this bacteria is going to behave as a pathogenic contamination, and if not, then it's effect on BE. The provided source notes reduced BE during their trials, however they admit that the reduction in performance was likely correlated to the reduction in spawn:
"although all treatments had lower BE in these experiments, perhaps because of the lower A. bisporus inoculation (0.7%) in both previous experiments, whereas it was double in our trial, i.e. we used 1.4% mycelia of A. bisporus spawning in the compost."
This seems like a serious mistake, why change an important variable half way through
I'm posting this before my results are in, in the hopes that others may be interested in running their own trials. My hope is to put a serious dent in seasonal trichoderma contamination.
More to follow...
Edited by Stipe-n Cap (01/12/21 09:28 AM)
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Josex
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27141948 - 01/11/21 12:35 PM (3 years, 17 days ago) |
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Thanks for sharing, will take a look later. There's nothing symbiotic about cube myc and bacillus though. Might be about the most disruptive bacteria there is for cubes.
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Josex]
#27141950 - 01/11/21 12:36 PM (3 years, 17 days ago) |
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Quote:
Josex said: Thanks for sharing, will take a look later. There's nothing symbiotic about cube myc and bacillus though. Might be about the most disruptive bacteria there is for cubes.
I think that depends on the strain of bacillus.
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Josex
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27141956 - 01/11/21 12:38 PM (3 years, 17 days ago) |
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Subtillis. The one that produces the endospores that we try to kill in the PC.
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Josex]
#27141971 - 01/11/21 12:44 PM (3 years, 17 days ago) |
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Yes, this is not just any bacillus subtilis however. We will see though. Perhaps you are correct, the thing is that we need to start adopting methods used in the agro community where applicable. Tests have not been done with active mycelium due to legal restrictions. I believe we are in a great Position as a community to be filling that niche.
It may be the case that this strain of bacteria is pathogenic to cubensis mycelium and not with agaricus, this is yet to be determined. It stands to reason however that if it's beneficial to one species, there may be others. Only way to find out is to noc up some jars and give it a go. If it works this could be a great breakthrough for hobby growers suffering from seasonal trichoderma. If it doesn't work, well, it will be fun to find out 
It is my intention to try as many bacterial inoculations as I can get my hands on. This is just a start.
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bw86
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142021 - 01/11/21 01:10 PM (3 years, 17 days ago) |
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So i have thought about this before. The fact that some fungi need a casing layer i think is helpful info to use. I have made post i will look for them in a little bit. From what i remember from my research is something called "Fire Fang" many people on the board who collect poo know what it is. Well its not just one bacteria. It's a plethora of micro organisms. Maybe we can get a lab (or bod ) to tell us what exactly is in it. Might be somewhere to start.
Edit - Oh yeah, I didn't read the post. Sorry I'll be back in here later.
Edited by bw86 (01/11/21 01:11 PM)
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starbones
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142027 - 01/11/21 01:15 PM (3 years, 17 days ago) |
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Just use t-methyl if you're worried about green molds.
You can look up the studies done and see the beneficial increase in BE when used as a casing drench. RR also used t-methyl to create grain spawn that didn't need to be PC'd but ultimately found it not to really be needed with proper sterile procedure.
I think t-methyl as a casing drench is now the industry standard for mushroom farms now? Ones that aren't looking for OMRI certification anyways. Keeps the green away except for t. aggressivum I believe which we probably don't run into much.
Senator 50 SC is one of the products you're looking for specifically designed for mold inhibition in mushroom farming. Instructions have a mix-rate, application rate etc. You won't see it used much here because of two factors, 1) Sterile procedure for spawn works 2) Clean spawn can take a sub to the 3rd flush without green mold.
There's also the problem of t-methyl being reaaaaally fucking expensive for most people as something like Senator 50 SC is a concentrate and you gotta buy a big jug of it. $1100 Canadian for one but in this game you'd never run out, ever.
Or just use clean spawn because sterile procedure isn't difficult and we're not using compost like white button farmers are so clean spawn + contamination resistant growing medium = green molds not really an issue until the myc is dying.
Hope that helps.
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Stipe-n Cap


Registered: 08/04/12
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: starbones]
#27142069 - 01/11/21 01:48 PM (3 years, 17 days ago) |
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Thats definitely interesting, post the particulars and that would definitely benefit the thread.
And yes, clean spawn blah blah, this is basic, standard mycology, it's a given. What I want is more tools in the chest. Trichoderma is a starting point. Learn about Japanese and Chinese growing practices, they utilize a wide variety of bacterial symbiots. This is what I want for actives. Trichoderma is a starting point. We are myopic,let's try not to be.
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mushhead
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142085 - 01/11/21 01:53 PM (3 years, 17 days ago) |
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 I have just the lab connections you're talking about. Guess I'm calling up all my microbiologist buddies and have them come over for a chat. EDIT: Shroomery on mobile sucks.
Edited by mushhead (01/11/21 01:54 PM)
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: mushhead]
#27142088 - 01/11/21 01:56 PM (3 years, 17 days ago) |
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Thats what I'm talking about. One day these species will be legal, let's do what we can to improve upon our understanding of the growth cycle and the beneficial relationships between fungi and bacteria. Things get monotonous, let's mix it up with some real science.
Edited by Stipe-n Cap (01/12/21 10:46 AM)
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Josex
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: bw86]
#27142098 - 01/11/21 01:59 PM (3 years, 17 days ago) |
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Quote:
I will be sourcing B. subtilis QST713 from Serenade antifungal spray to begin my trials. My intention is to inoculate petri dishes with B. subtilis QST713 using an inoculation loop and then transferring bacteria colonies to LC; LC will then be used to coat grain spawn.
I grow cubes and tbh I don't know anything about aggro applications of B. subtilis. All I know is that the last thing I want in my spawn is B. subtilis, that's why we go to great lengths to destroy its endospores on our grains.
It is a very common contaminant of grain spawn, more so than we give it credit for and even slighly contaminated spawn can and will reduce yields by a lot. There's very little information on the subject of B. Subtilis in regards to mushroom cultivation (cubes) and I'm not an avid reader on the subject myself but from my own experience I have linked the presence of this bacteria in my spawn to a number of serious issues in my tubs that may go beyond mere correlations.
B. subtilis also produces cry toxins when they produce their spores, a powerful antifungal, and a subset of the population produces spores even in the presence of abundant food and good conditions, so if there's B. subtilis in yous spawn you can be sure it will produce spores (and therefore cry toxins) when spawned.
So no need to source anything. Look no further than the grain you use, as it's naturally loaded with B. subtilis spores.
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Josex]
#27142106 - 01/11/21 02:04 PM (3 years, 17 days ago) |
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There will be detractors, certainly. This happens any time that someone thinks outside of the box. Bacillus is a contaminate species for all mushrooms. They developed this strain by isolating bacteria that clings to the mycelial plane of certain species of gourmet mushroom. Unless you have imperial data proving otherwise you cannot say with any certainty that it will not benefit cubensis.
Bacterial symbiosis is a thing for all species of fungus, including cubensis. We will see.
Saying: "Bro, clean spawn" isn't helpful. The gourmet industry does what it does for a reason.
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Josex
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142114 - 01/11/21 02:06 PM (3 years, 17 days ago) |
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Josex]
#27142118 - 01/11/21 02:08 PM (3 years, 17 days ago) |
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Listen man, you're a guy who grows illegal drugs, not a real mycologist. Face palm me all you like.
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Josex
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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142129 - 01/11/21 02:13 PM (3 years, 17 days ago) |
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All I'm saying is, if you want B. Subtilis because you think is some hot shit for growing cubes, look no further than your own grains. All you need to do is an insufficient cycle and you will have a lot of B. subtilis in your spawn, look for tha nice wet spot.
Then spawn that and you'll see some symbiotic action from hell take place. 
You only need axenic spawn to produce healthy good flushes, no need to add anything or spray anything to your substrate and certainly no bacteria ffs.
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Josex]
#27142145 - 01/11/21 02:23 PM (3 years, 17 days ago) |
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Yeah, I agree 100%
What I'm saying is that is 100% basic information provided to every noob ad nauseum. This is fucking boring. I can make clean spawn with my eyes burned out with a cigarette.
I want to do some science, this is how it's done. I'm not looking for a silver bullet, what I am looking for is an advanced approach to understanding the symbiosis between active species and bacteria, discount as you may, there certainly is one, or else they would not have evolved on dung, or at all.
Problem:
Seasonal trichoderma plagues hobby active mushroom growing community.
Hypothesis: By Understanding symbiotic relationships between cultivated fungi and bacteria we may benefit the community and deepen our understanding of the species we work with.
You are correct, this may fail, but it may not. If you're content growing cubes ad infinitum then keep doing that. I on the the other hand require a tad more intellectual stimulation.
This is a fun project, I'm sharing it. If you think that clean spawn and sterile technique are the depth and breadth of our trade, so be it. I on the other hand would be far more interested to find more.
Edited by Stipe-n Cap (01/11/21 02:38 PM)
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Lemgrub



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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Stipe-n Cap]
#27142148 - 01/11/21 02:25 PM (3 years, 17 days ago) |
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There's probably some bacteria with mutualistic relationships with cubensis. Actually there almost definitely is. If I were looking for them, I'd start with their historical growth medium, namely shit. Find out the main members of the microbial community in a range of samples from a few different species of animal and try to find recurrent species of bacteria.
I think this type of addition would just be redundant to the best growers here as they get very high BE, but it may have merit for the less experienced grower.
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Stipe-n Cap


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Re: Axenic inoculation versus symbiotic bacterial inoculation [Re: Lemgrub]
#27142155 - 01/11/21 02:26 PM (3 years, 17 days ago) |
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Like I said, if good enough is good enough for you, well, good.
Cubes aren't the focus here, just to be clear. Why do pans need manure? Almost certainly because of bacteria. Cubes, pans, whatever species you care to name will most certainly have formed a symbiotic relationship with bacteria and I am going to try to find out which ones. My starting point is logical, I'll start with known species of bacteria used within the gourmet and agro community, if they fail, well, I'll keep looking.
Yes, you can grow a shit ton of mushrooms, but is that what makes you happy? If it does, good, I on the otherhand have a "lab" and I certainly intend to max out its potential.
So, why bother saying "why bother"? it is what it is. This threads purpose is to experiment with inoculation procedures that venture outside the norm, is it necessary for growing mushrooms? Big nope. Is it fucking cool to think and experiment? Resounding yes.
Even if I fail in my attempts, I have still found an answer.
Edited by Stipe-n Cap (01/11/21 02:46 PM)
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