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InvisibleCreonAntigone
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Registered: 05/30/21
Posts: 2,875
CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER * 1
    #28133169 - 01/10/23 05:32 AM (1 year, 17 days ago)

A. INTROUDCTION: MOTIVATION FROM A CURIOUS AND FASCINATING RESULT. Adding a culture = less contaminants?

After a rather haphazard experiment, I achieved a result that amazed me. I was working with a culture of P. Cubensis and had made a large transfer to this 'plate', which was a jar that was filled with media and sterilized, as a 'no pour agar'. Media composition was a standard MEA recipe I found. As the mycelium grew, I also noticed the growing of a penicillium mold contaminant. Here it is early on in the growth.





Several weeks later the penicillium mold had grown as had the mycelium. This was not too big of a concern as there were many areas I could transfer from to get a clean culture. However, I decided to take an unconventional approach. Instead of taking a culture away, I added a culture. I poured about 1.5ml liquid culture of Tuber Melansoporum, a mycorrhizal tree endophyte that produces fruit bodies known as 'black winter truffles'. The fruit bodies take years to form, but in its mycelial form, it is a symbiote. In addition to its form as a tree symbiote, clones of fruitbodies can grow out in pure culture, although they'd never fruit in such conditions. I had been working with several clones of it in LC, and I wanted to see what would happen when it was grown on a plate that mushrooms were already growing on.

The results of that experiment are here. What surprise me the most is that the contaminant disappeared and is no longer there. I presume that the truffle mycelium allied with the mushroom mycelium and helped it defeat the penicillium mold. This may be strange because truffles have no interaction with P. Cubensis in the wild; yet truffles do cooperate with mushrooms in the wild (as certain mushrooms like boletes are known to appear more in truffle regions). I presume this result obtains because truffles are capable of forming at least preliminary symbiotic relationships with many different kinds of species, not just trees, and if two fungi are put together, they might start cooperating in ways that would be impossible outside of controlled conditions.





What are we seeing here? It looks superficially at the top like mushroom mycelium, and you might suppose that maybe the mushroom just won control of the culture; but look closer. In the next picture, we can see black dots growing within the myc like a network. I suppose these are the sclerotia of black truffle. While it would not fruit under such conditions, I have seen sclerotia of T. Melanosporum in many saprophytic cultures.



And, what is most interesting is the bottom of the culture - it is growing down, producing white-to-black mycelium that has a different texture than typical mushroom myc. I have done these 'no pour agar' in jars before for mushroom myc, and it typically does not grow down, while truffle species do. The only cultures in which I saw it grow on the side of the jar and down the culture like that were my agar plates of my truffle clones.

My theory is this culture represents a mycorrhizal co-culture. I don't have proof of this, nor a microscope to confirm it. I am sure people here might come up with alternative explanations for why what I'm seeing is not the thing I'm thinking it is. That's possible. I'll put a pin in that, and conduct two new experiments.

EXPERIMENT A. FRUITING THIS CULTURE

I plan to see if I can grow mushrooms using transfers from this agar plate. I will probably use violet's pods tek and attempt to fruit it within self-contained jars. I will update this section as I prep some grains and transfer.

I expect the mushroom myc to grow similarly, but what would be interesting is whether we might see sclerotia of the Tuber species (again we won't see fruits in such a condition, but sclerotia seems possible). If I get sclerotia, it can't be from the mushroom, since it is P. Cubensis and not a sclerotia-forming mushroom, and I will presume they are the product of the truffle. I will send in any sclerotia to get sequenced if they do end up growing.

EXPERIMENT B. CO-CULTURE AS CULTURE DOCTOR, ATTEMPTING A NEW MYCORRHIZATION: P. SEMILANCEATA PLUS T. MAGNATUM


What interested me about the past experiment was the addition of the truffle species seemed to act as a 'doctor' for the mushroom: it removed the penicillium contaminant and allowed it to dominate the plate.

So, in this experiment I will add my best truffle culture to a mushroom culture that is doing extremely poorly, in the hopes to see whether the truffle might ally with the mushroom and help it beat the contaminant, as I theorized earlier. I expect a lot of skepticism as to this part of the experiment, and I have some myself. A failure may not mean it will fail in every case, it might just mean this culture in particular is too far gone. But I'm curious to see - if the myc gets help even a little after the addition of the truffle, but especially to the point where the culture seems usable, I will be very intrigued.

Therefore this experiment was already ran, and I will describe it:

I have two cultures that started P. Semilanceata spores on a rye flour media, as I describe in this post. One culture is doing well; it has some orange metabolites likely indicating bacteria, but the mycelium is nonetheless consolidating, and I will likely with transfers be able to get a pure culture:



On the other hand, one of the cultures is very sick. There are sections of mycelium that looks okay, most is multi-colored molds.



This experiment consisted of adding truffle liquid culture, about 5ml, to the diseased culture, in the hopes of seeing if - as in the example of OP in which the truffle appears to have eliminated the contaminant - the truffle can ally with the mushroom and act as a 'doctor' and cure the sick culture and make it useable. You can see that the culture is in a very poor condition.

I started this experiment 2 days ago. I added 5ml of my best truffle liquid culture, which was a Tuber Magnatum (white truffle) LC growing on sterilized honey, which I isolated myself from a fruitbody clone. I want to see how this truffle culture interacts at being thrown into such a harsh environment.

This experiment is a bit more 'off the wall' and less promising than the experiment A, since the culture involved is in such bad shape. But nonetheless I want to see the results.

CONCLUSION: WHY THIS EXPERIMENT?

We know conclusively that many mushrooms grow, such as P. Cubensis, grow just fine in pure culture. So why would anyone want to introduce a symbiotic fungus into the culture, what purpose would it serve?

It could see this being potentially useful in two circumstances: first if there is a culture that is very contaminated and efforts to fix it haven't helped, attempting a new symbiotic culture might be a method to help the mycelium grow in a way it could not before. Second, for species that haven't proven as easy to grow as P. Cubensis, such as P. Semilanceata, growing along with other species may hold a key to increasing the likelihood of fruiting.

I don't expect it to be hard to grow out and fruit the cubensis culture. I am mostly curious to see if it retains any evidence of being a mycorrhizal culture once fruits appear.


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Invisiblebakedbeings
orbiter of truth
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Registered: 09/01/20
Posts: 4,218
Re: CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER [Re: CreonAntigone]
    #28136355 - 01/12/23 08:25 AM (1 year, 15 days ago)

:popcorn:


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Offlinekedi
people are strange


Registered: 01/14/23
Posts: 48
Last seen: 7 months, 7 days
Re: CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER [Re: bakedbeings]
    #28146464 - 01/18/23 07:05 PM (1 year, 9 days ago)

yeah thats cool... my thınkıng ıs that ıt would be competıtıve.. I trıed googlıng mushroom to mushroom symbiosis wıth no luck.. are there other examples of mycorrhızal symbıosıs between mushroom specıes?

ım ınterested ın growıng out Melansoporum on graın and lc and addıng methoınıne for sulfur compounds.. do tubers act lıke regular mycelıa? you mentıoned downward bıts.. any advice? I also read that lıme somehow worked to get the trees establıshed..


Edited by kedi (01/18/23 07:08 PM)


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InvisibleCreonAntigone
Stranger

Registered: 05/30/21
Posts: 2,875
Re: CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER [Re: kedi]
    #28146773 - 01/18/23 10:50 PM (1 year, 8 days ago)

Quote:

kedi said:

ım ınterested ın growıng out Melansoporum on graın and lc and addıng methoınıne for sulfur compounds.. do tubers act lıke regular mycelıa? you mentıoned downward bıts.. any advice? I also read that lıme somehow worked to get the trees establıshed..




Grain is not the best idea, as they (Tubers) lack the ability to really break down materials unlike a mushroom. A flour cake might work. But start with simple sugar media, I suggest agar to LC on malt extract before trying a cake. I tried whole grains and they didn't seem to work as the mycelium can't really colonize in the same manner as mushrooms.

Limes purpose is for adjusting soil pH to mimic natural truffle grounds.


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Offlinekedi
people are strange


Registered: 01/14/23
Posts: 48
Last seen: 7 months, 7 days
Re: CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER [Re: CreonAntigone]
    #28147829 - 01/19/23 04:45 PM (1 year, 8 days ago)

ya i did some reading and found soybean and red adlay flour were tested for solid-state media. 1:3 ratio was the best for biomass and antioxidant activity..
https://pdfs.semanticscholar.org/d010/012122d476f08f046e6732cdb320f0a90ae8.pdf

also https://www.nature.com/articles/s41598-018-27620-w
was about introducing methoinine.. but i think in this one they suggest sucrose which seems weird.. but .. truffles..

but yes agar agar agar agar ...

thanks


Edited by kedi (01/19/23 04:50 PM)


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Offlinekedi
people are strange


Registered: 01/14/23
Posts: 48
Last seen: 7 months, 7 days
Re: CO-CULUTRE EXPERIMENT: MYCORRHIZAL SPECIES AND MUSHROOMS TOGETHER [Re: kedi]
    #28152602 - 01/22/23 06:15 PM (1 year, 5 days ago)

oh and I thought maybe the yeast around the tuber could be symbiotic, but my guess is more of a benign mutualism


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