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Offlinedschwar2
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Oregon Black Truffle (Leucangium carthusianum) culture parameters * 2
    #28370250 - 06/22/23 02:01 PM (7 months, 2 days ago)

The basic method of truffle cultivation consists of inoculating a tree seedlings roots with truffle mycelium or spores and then waiting for the tree to produce truffles. Also, planting seedlings in the vicinity of a known truffle producer, or using sections of infected root. Most methods on how to actually inoculate the roots describe using a spore slurry. Culturing on agar/LC is supposedly challenging and there’s very little information on it. I am trying to culture it on agar and hopefully pinpoint some parameters for producing liquid culture which can be used to more efficiently inoculate seedlings vs the spore slurry method. Since I am based in Oregon, I’ll be trying the Oregon Black Truffle (Leucangium carthusianum).

I started with several freshly picked truffles (forgot to take photos of these but they were around the size of golf balls). I wiped down the edges with isopropyl, then peeled the skin off one side with a scalpel to expose the interior. I then cut wedges and transferred to the agar plates I prepared. I made a total of 5 plates. I probably should have done more, but didn't expect anything to happen and didn't want to waste the plates. I tried to cut fairly small pieces, since I was worried about contamination from the exterior of the truffle. One piece of tissue I cut fairly big and that was the only one that grew out.


Clone plate with no growth


Clone plate with no growth (the specks are condensation)


For the clone plates, I used standard MEA- no yeast or peptone. I didn't PH adjust. Most gourmet European truffles like a PH of around 8, as they grow in alkaline calcareous soils and the few papers I've read trying to culture European truffles all suggest PH adjustment of media. Since L. carthusianum is known to exclusively colonize Doug Fir trees in black acidic soils in the PNW, I decided not to change the PH.

I kept the plates (started 4/7/23) at room temperature for several weeks. I didn't see any growth, so I transferred them to the fridge. I forgot about them until the end of May, and found that one plate was showing growth.

Clone plate with growth (front) radiating out from transfer. This is after I took some to transfer to new plates.


Clone plate with growth (back) radiating out from transfer


From that plate, I transferred several wedges to new dishes. These were MEA-Y plates I had already poured for my woodlovers using alder soak water, a small amount of peroxide, and blue food coloring.

After several days, these plates began to show growth as well (albeit somewhat wispy).











Rewind to the start of April. I had also blended a peeled truffle in sterile water with a very small amount of LME. I had originally intended on using it to inoculate some Doug Fir seedlings but had other things going on so the solution sat in my fridge for two months. When I checked it at the end of May, it looked like a typical liquid culture, with uniform growth and no strange colors or sediment. I was somewhat skeptical and so I transferred a few drops of it to one of the aforementioned MEA-Y peroxide dishes. The solution moved all over the plate so if it was heavily bacterial one would expect colonies to pop up all over the place. Instead, it grew in one mass right where I pipetted the liquid. This plate looked more similar to the initial clone plate I made, with off white dense growth vs. wispier mycelial growth which I got from the second transfer. My guess is there could be two different kinds of growth happening here- mycelial vs fruit body growth for lack of better terms.


Liquid culture plate (back)


Liquid culture plate (front)...I ran out of agar


Liquid culture plate (front)

Going forward, I'd like to make some more transfers to see if I can get some different growth. I'm going to experiment with PDA-Y as I think the starches will be more similar to the plant starches the truffles get from their host. I am also going to try to expand the LC I have so I can spray around the small Doug Firs in my yard. Lastly, I'm going to scope out the plates under a microscope and take some pictures. I think the easiest method for producing Doug Fir seedlings is to germinate seeds in a sterile petri dish and then immediately transfer the sprouts to colonized agar plates for a day or two. Then scrape up the mycelium on the surface with the sprout, and transfer to some sterile soilless media impregnated with more liquid culture. Then periodically give the seedlings more LC.

If anyone has any suggestions on nutrient recipes/next steps please let me know.


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InvisibleCreonAntigone
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: dschwar2]
    #28370324 - 06/22/23 03:17 PM (7 months, 2 days ago)

My recommendation is that you don't wipe them down with isopropyl, and take selections for cloning that include the outside of the truffle. The reason is, truffle species such as this are a whole microbiological community - just like how they grow in connection with trees, they grow in connection with yeasts and bacteria. The hard outer edge of the truffle is composed primarily of yeasts and bacterias that are allied with the truffle, and these you want to culture. You can culture without them, but the culture will go much much slower if you don't get these commensural bacteria.

EXAMPLE - in T. Borchii (it's in the same family but not the same genus), the smell is by this study said to be entirely produced by commensural bacteria.

This article says the aroma is probably made by microbes for all truffle species - some made by the fungus, most by bacteria.

But the research has found that the outer edge of the truffle, the peridium, has an entirely different community of bacteria - as demonstrated by this study, which finds about double the number of bacteria geniuses in the peridium than in the gleba of T. Melanosporum. And you WANT to cultivate these bacteria too, because  the bacteria help the truffle colonize (everywhere the bacteria colonizes, you'll see mycelium too - the bacteria and the truffle exist in a mycohrizal arrangement.)

I think the most efficiacious and quickest method would actually be starting in LC, from a fruit body piece. This is highly inadvisable in mushrooms, but I think it is the best for truffles, for this reason: truffles want a bacteria-rich and yeast-rich environment, as liquid culture is if you inclue the outside of the fruit. The truffle will grow faster the more 'contaminated' the culture is, so long as the contamination comes from the truffle itself and the natural allies that are already in the fruit bodies.

My suggestion would be for your next culture, make sure to include a piece of the outside of the fruit body, after washing. What you can even do is leave the truffle in a container with a piece of paper towel in the fridge and wait for it form mycelium on the outside, which it does. Taking it from the inside would work too, but just worse in my eyes. If it relies on these yeasts and bacteria to fruit in nature, a culutre without it would be less vigorous... it would grow less fast, be less resistant to contamination (because it no longer contains its native yeasts, so is vulnerable to non-native ones), in short, it's worse in most ways in my eyes.

I really do suggest a 'pepsi challenge' here and try two cultures side by side, one including a lot of the outside edge, the other not. You can even have one plate that uses all the outer edges, and compare it to these plates with internal portions. I think your growth will be paradoxically much 'cleaner' if the native yeasts are allowed.


Quote:

dschwar2 said:






Looks like penicillium to me.. is the mycelium only looking like that due to the blue dye of the agar, or should it still look white? But I see blue fruits inside a white colony, that's a dead giveaway for penicillium I think.


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InvisibleCreonAntigone
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: CreonAntigone]
    #28370403 - 06/22/23 04:37 PM (7 months, 2 days ago)

Oh, also -
Quote:

Most methods on how to actually inoculate the roots describe using a spore slurry. Culturing on agar/LC is supposedly challenging and there’s very little information on it.




New studies have now proven that isolated culture inoculation can inoculate trees that are productive and make truffles. Try this study on for size.

First evidence for truffle production from plants inoculated with mycelial pure cultures (2016)
Mirco Iotti, Federica Piattoni, Pamela Leonardi, Ian R. Hall & Alessandra Zambonelli

Quote:

Truffle (Tuber spp.) cultivation is based on raising mycorrhizal trees in greenhouses that have been inoculated with suspensions of ascospores. The problem with this is that pests, pathogens, and other mycorrhizal fungi can contaminate the trees. Furthermore, because ascospores are produced sexually, each plant potentially has a different genetic mycorrhizal makeup from each other so tailoring the mycorrhizal component of plants to suit a particular set of soil and climatic conditions is out of the question. Here, we report on the production of Tuber borchii-mycorrhized plants using pure cultures, establishing a truffière with these and subsequent production of its fruiting bodies. This study opens up the possibility of producing commercial numbers of Tuber-mycorrhized trees for truffle cultivation using mycelial inoculation techniques. It also poses questions about the mechanism of fertilization between the different strains which were located in different parts of the experimental truffière.





Article full text (obtained via sci-hub)


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Offlinedschwar2
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: CreonAntigone]
    #28370583 - 06/22/23 07:30 PM (7 months, 2 days ago)

Thanks for your response! What you say about the commensural yeast and bacteria is an interesting point. Thanks for the article, it seems like most of the literature out there is using T. borchii. There being less yeast/bacteria in the gleba than peridium makes sense, since the peridium is in contact with soil microbes and has to compete with them. I only did a quick wipe with the isopropyl to get dirt off but I'll have to try one with skin on, no isopropyl.

Random thought- I wonder if some of those commensural microbes would outcompete with the mycelium if in too high of concentrations or on agar. Because you're basically changing the entire environment- no roots, no soil, excessive nutrients that are not usually there. Sort of like humans can have flora overgrowth where beneficial microbes become harmful when there's too much sugar or immunocompromised. It sounds like this is not the case though.

It's definitely not penicillium, or any mold I've seen. Too slow growing and too dense. These plates are all three weeks old. The one you refer to is from the LC, and it added way too much moisture into the dish. I used a dangerous amount of blue food coloring to the point where the condensation was blue, so the added moisture condensed on top of the mycelium and stained it. It's difficult to see. If anything, to me it looks somewhat bacterial. I don't think it is bacteria, I think it's just the weird looking truffle mycelium that's excessively wet. Could be wrong though.


Here's a drier one for reference.





Liquid culture plate (back)

The truffle I used for the LC was pretty crudely peeled. I intended on using it outside as a spore slurry and not as an LC. That could be why it grew so well. I plan on transferring some to new plates tonight to see if it grows differently without all that moisture.

I'll probably be working with the cultures I have for the near future as I don't have access to them right now and they're expensive to buy.


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InvisibleCreonAntigone
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: dschwar2]
    #28370745 - 06/22/23 10:36 PM (7 months, 1 day ago)

Ah yes, blue condensation makes sense. In that case both cultures look pretty good.

I may not be the best mycologist, you can take my ideas with a grain of salt - however, I can say I'm one of the few on this forum that has made any attempts at trying truffles (albeit they were adhoc and experimental). I've worked on truffles for 2 years, and currently have some LC and some plates growing from some T. Aestivum that I got this season. I did them with 'no pour' agar in jars, so they're hard to get pictures of, but I'll get some clearer containers to transfer to and post them here at some point.

My suggestion to you is, put your saplings in a controlled environment - such as a greenhouse or a small humidity chamber (eg a tub with wet soil, but make sure it has been well-drained after hydrating). Propagate one of your cultures in a large batch of LC then regularly spray the leaves of the plant with LC.

Try higher nutrient LC, like 10-20% nutrients - truffles seem to grow faster in that, and it also forms mycelium structures that sometimes coat the top of the medium.

One thing to be aware of is that even though truffles are symbionts, one can have 'too much'. A sapling that has been loaded down with spores will have its leaves turn black but will continue to stand in the soil. When this happens, though the tree is dead, the sapling is coated in asexual spores and becomes sort of like an infected stick. So all one should do is try and plant another sapling nearby, and the truffle spores will passively travel via insects to the other new sapling, but it will be less likely to due because the last infected tree will offer some resistance.

This was the key lesson I want to impart, that mycorization may require iterated interactions between truffle and tree. My example is, I tried infecting a quaking aspen tree with summer truffle, and wanted to start with small saplings. I took rooted shoots and, in pots in a room in my basement that served as a greenhouse, watered them with spore slurry. They (over the course of 4 weeks) turned black, and the soil took on a black character too. However, I left them for a few months (watering occasionally). A few of the trees which I thought were dead, weren't. Some regrew leaves. And I could tell that even though a few of the trees had died (some had not), truffle spores seemed to have coated the top of the soil in these little pots.

I tried again a year later,  quaking aspen saplings again, in the same room. This time I used soil derived from the soil I'd prevous exposed to truffles. And this time, I also started from cuttings and rooted them in the soil. This time, miraculously, the cuttings survived and thrived. I sprayed them with liquid culture, and added a new truffle fruit body to the soil, and they did not react negatively at all. I had to move but they were growing flowers right as I moved - even as I exposed them to more truffle spores. It was a rate of 11/11 successfully rooted cuttings, higher than what typically happens with cuttings.

My conclusion from this experience is that even if truffles can infect a plant a bit too much, the next plant in the same soil will have a certain resistance to it and won't immediate succumb, and as a result it will be possible for mycorrhizae to form. Iterated interactions. In some sense there's a repeated learning, the truffle learning to infect the tree, but also the tree learning not to get over-infected and die.


Edited by CreonAntigone (06/22/23 11:03 PM)


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Offlinedschwar2
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: CreonAntigone]
    #28385309 - 07/04/23 07:12 PM (6 months, 21 days ago)

Yeah I'm surprised more people on here don't try truffles. I bought several bare root Doug Firs that I inoculated and they all died. I think they were just stressed from getting shipped. I also inoculated a couple 1-2 year old nursery bought Doug Firs and they're planted in my yard and thriving. I now have three seedlings that sprouted around my yard which I dug up, soaked them in distilled water, and then scraped mycelium from a plate onto their roots. Then put them in solo cups with perlite, vermiculite, peat, and coco coir. They're all doing pretty well so far.

Interesting about the LC. You use LME for the liquid culture? And do you add peptone or yeast extract as well? I'll try a higher percentage of sugars.

Also, why spray the leaves vs drench the roots? Seems like they'd possibly mold easier if there was sugar on their leaves?


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InvisibleCreonAntigone
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: dschwar2]
    #28392214 - 07/11/23 08:31 AM (6 months, 14 days ago)

The principle behind spraying the leaves would be: quicker and easier invasion of the truffle into the plant. There would be some residual sugar but most would be consumed as the culture grew; mostly what you'd be distributing on the plant is live mycelium.

I think spraying the leaves would be more direct and effecient because it applies to the plant directly, as opposed to in the soil where it might not be directly touching the plant. It's possible though that touching a root is faster than touching a leaf, and so your method may turn out to be better.

As I said too much culture at once could be detrimental and lead a sapling to die. So my thought was a very light misting, like a spray bottle squirt every week or so, repeated applications of a small amount. That wouldn't add an excess of sugars, and a low mix could be used for this in particular.

Quote:

Then put them in solo cups with perlite, vermiculite, peat, and coco coir




I'd suggest mixing in some native soil. Native soil would acclimate your saplings to grow in the area you'll eventually plant them in.

I don't know about peat in the mix, it may be a bit too acidic even for Douglas fir. I think as opposed to with saproptrophic mushrooms where you want to get one species on its own, if you're growing a mycorrhizal species you might want to emphasize the interaction between the target fungi and surrounding species. By that principle live soil and compost could be the best growing medium for this kind of project.

Quote:

Interesting about the LC. You use LME for the liquid culture? And do you add peptone or yeast extract as well? I'll try a higher percentage of sugars.





I use dark malt syrup, shouldn't perform any different than LME.

I'm not sure about those additives, you could try them.

Some research has been done on additives for truffle fermentation. I'll find articles later.


Edited by CreonAntigone (07/11/23 08:39 AM)


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Offlinedschwar2
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Re: Oregon Black Truffle (Leucangium carthusianum) culture parameters [Re: CreonAntigone]
    #28408130 - 07/25/23 04:05 PM (6 months, 6 hours ago)

Quote:

CreonAntigone said:
The principle behind spraying the leaves would be: quicker and easier invasion of the truffle into the plant. There would be some residual sugar but most would be consumed as the culture grew; mostly what you'd be distributing on the plant is live mycelium.





Right, but the truffle mycelium is ectomycorrhizal and is only known to associate with the root system of the Doug Fir. The needles aren't going to be able to absorb the mycelium and transport it through the phloem to the roots. Seems to me like if the goal is to colonize the roots, root inoculation is the most direct route. I've just been injecting LC directly into the root zone of my seedlings and they are looking very healthy.



Quote:

CreonAntigone said:
I'd suggest mixing in some native soil. Native soil would acclimate your saplings to grow in the area you'll eventually plant them in.





I plan to start incorporating native soil once they get bigger. I think that starting them in a clean soilless medium (i.e. not tons of other competing mycorrizae or thrichoderma that will eat it up before it can colonize the roots) will allow the truffle mycelium to gain dominance as more and more root tips develop before exposing it to competition. I've planted nursery grown Doug Firs that spent their first two years in just bark mulch and slow release fertilizer and as long as they are planted in the fall and don't get drought stressed, they acclimate well to the soil.

Quote:

CreonAntigone said:
I don't know about peat in the mix, it may be a bit too acidic even for Douglas fir.
I think as opposed to with saproptrophic mushrooms where you want to get one species on its own, if you're growing a mycorrhizal species you might want to emphasize the interaction between the target fungi and surrounding species. By that principle live soil and compost could be the best growing medium for this kind of project.





The peat could be a little on the acidic side. I really use it sparingly. Mostly vermiculite, perlite and coir. Plus Oregon Black truffles and Doug Firs grow in super acidic soil, unlike other truffles so I don't think it'd be a huge issue. Maybe something to investigate further.
I feel like you may be overemphasizing the interaction between the truffle mycelium and other microorganisms. The primary goal/interaction I am looking for is between the rhizosphere of the Doug Fir and the truffle mycelium. Native yeasts/bacterias will naturally co-colonize the growing medium if I keep them in free air. What I don't want is fungal competition. Not until the root system is developed and has been sufficiently colonized by the truffle mycelium. I have one mature Doug Fir (~40ft tall) and all of my neighbors have Doug Firs as well, so I can assume they are all colonized by other ectomycorrizae that will be present in the soil and compete with the black truffle mycelium. I do think the native soil microorganisms are important for fruit body development but are less important for mycelial growth and root tip colonization.
I'm just talking out of my ass now but morels form sclerotia when exposed to stresses/dessication/native soil and those sclerotia can then turn into fruiting bodies. Leucangium is actually part of the Morchellaceae family, so could also form fruiting bodies similarly and be dependent on the same kind kind of stimuli. Morel mycelium can easily be grown without any other organisms, the big unknown is when you try to fruit the mycelium.

Quote:

CreonAntigone said:
I use dark malt syrup, shouldn't perform any different than LME.

I'm not sure about those additives, you could try them.

Some research has been done on additives for truffle fermentation. I'll find articles later.




I made some LC with ~8% LME and a knife tip each of peptone and yeast extract. Added a few wedges of mycelium on agar and it grew out pretty aggressively in the solution. I'd like to figure out a better agar recipe for it though. May try using 2x nutrients in the agar as well.


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