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Anonymous

Rhizomorphic and "cottony" mycelia
    #2461381 - 03/22/04 09:02 PM (13 years, 3 months ago)

I personally have never done agar work, so I am unfamilar with the practise of agar work. I do have a few books that I've poured over and come up with a few questions. Most of these are based on the writings of Stamets in TMC.

Stamets talks about growing multispore on agar. He talks about isolating a rhizomorphic section of the mycelia. Now, if a rhizomorphic section is taken, blended with sterile water, and loaded into syringes that were used to knock up grain jars, would it eventually "degrade" in some way that would allow cottony sectors of mycelia to grow?

I guess what I'm trying to ask is how does mycelia go from rhizomorphic, and clones of rhizomorphic "strands", to becoming cottony again? Does the DNA in the mycelia "tell" it to form this undesirable texture? If so, does it work the other way around? Would a cottony sector, grown out in agar plates to a power of 100, eventually display very rhizomorphic tendancies?

I hope this makes sense, thx.


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Offlinethenetherworld
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2461583 - 03/22/04 10:06 PM (13 years, 3 months ago)

I think this is a well thought out question, now would one of the board experts explain this to us?


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Invisibletahoe
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2461764 - 03/22/04 10:57 PM (13 years, 3 months ago)

if it degrades it will be after a few cycle, The best thing to do with this is use that syringe with your isolated mycelium as an inoculant for another petri and use the rest on jars. If its a pimp strain then you can just take from the petri instead of the clones. it is much easier to keep a petri sterile then a shroom. I know this isnt exactly answering your question but i have dont what you are thinkg about and it isnt worht the time


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I need to proofread


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Offlinestarspawn2
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Re: Rhizomorphic and "cottony" mycelia [Re: tahoe]
    #2462803 - 03/23/04 04:31 AM (13 years, 3 months ago)

I don't know the facts of why the mycelium changes,i think starch from whole grains causes it to happen.I started mixing corn cob litter in with millet,cracked corn,and bird seed mix.The thing i started seeing was the mycelium reaching over the corncob to a piece of grain.The rhizoids were very large and connected over and in the corncobs.I have also cased single jars that looked very cottony only to find that when it colonized the casing it became rhizomorphic and fruited normally.I think the lack of nutrition might be what causes it.Age is a factor but its not the only one.


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: starspawn2]
    #2464693 - 03/23/04 07:33 PM (13 years, 3 months ago)

I don't think I was totally clear in how i asked the question. Let me do some examples.

1) If I have an agar dish that I multisporred, and I take a section of the rhizo strands and inoc OTHER agar trays with it, and constantly keep doing that, will I ever end up with cottony mycelia? If so, how many times. I think that the best way to express this asnwer would be in number of mass multiplications that need to take place for this to occur.

2) If the mycelia in the above statment will eventually turn cottony, wouldn't a liquid mycelia injection into a grain jar (or even "worse", a bag) cause the hundredfold or so multiplications in size that would lead to cottony mycelia developing again?

And now, down to the practical. If one gets rhizo mycelia from an agar plate and blends with sterile water, then inocs, say, 10 jars with it, and uses each of those 10 jars to inoculate 10x the mass ofthe jars in spawn, that seems like a hundred-thousand-fold replication in size and in more mycelia. Does this mean that starting off with a GREAT looking rhizo section of mycelia would, by the end of the multiplication of tissue, end up as whatever the "natural average" is for rhizo and cottony sectors?


Again, sorry if I didn't word this right.


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OfflineErik006
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2464774 - 03/23/04 07:55 PM (13 years, 3 months ago)

>1) If I have an agar dish that I multisporred, and I take a section  of the rhizo strands and inoc OTHER agar trays with it, and constantly keep doing that, will I ever end up with cottony mycelia? If so, how many times. I think that the best way to express this asnwer would be in number of mass multiplications that need to take place for this to occur.

First of all its multispored :wink:
Yes, you will most likely end up with cottony mycelium in the end, but what you are talking about is pure strain isolation,  in which the goal is to single out the best mycelium, once you have done that, you a have a aquired a pure (SUB?)strain which stamets calls p1 I believe, every transfer from this plate will result into a new generation, p2 respectively. (unless you keep your old culture) Now, in theory, you could keep going for quite a while.
But contamns are always sneaking up on you.

A few factors that increase the lifetime of your cultures
-Using different agar formula
-Isolation of only the best looking mycelia
-Reisolations from multispore inoculations
-Cold storage of your culture.

Maybe someone can help me out with a number here?

>2) If the mycelia in the above statment will eventually turn cottony, wouldn't a liquid mycelia injection into a grain jar (or even "worse", a bag) cause the hundredfold or so multiplications in size that would lead to cottony mycelia developing again?

I take it you mean rhizomorphic mycelia would grow from the cottony, and although im not 100% sure on this, I would have to say no, because since you have a pure culture, meaning the mycelium is identical, and the strain has degraded, the newly grown mycelium will also be cottony. This is when you would isolate a new strain from multispore inoculation.

>And now, down to the practical. If one gets rhizo mycelia from an agar plate and blends with sterile water, then inocs, say, 10 jars with it, and uses each of those 10 jars to inoculate 10x the mass ofthe jars in spawn, that seems like a hundred-thousand-fold replication in size and in more mycelia. Does this mean that starting off with a GREAT looking rhizo section of mycelia would, by the end of the multiplication of tissue, end up as whatever the "natural average" is for rhizo and cottony sectors?

No, the strain you isolated is rhizomorphic, if your strain has not degraded yet, the new growth will be rhizomorphic too.

Erik006


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: Erik006]
    #2464843 - 03/23/04 08:20 PM (13 years, 3 months ago)

Quote:

Erik006 said:
I take it you mean rhizomorphic mycelia would grow from the cottony, and although im not 100% sure on this, I would have to say no, because since you have a pure culture, meaning the mycelium is identical, and the strain has degraded, the newly grown mycelium will also be cottony. This is when you would isolate a new strain from multispore inoculation.




So if I started off with a strain of rhizomorphic mycelia, as long as I kept using that, or the "children" of that, I'd always have rhizomorphic myeclia? Thats not what stamets says, I think
Quote:


No, the strain you isolated is rhizomorphic, if your strain has not degraded yet, the new growth will be rhizomorphic too.




What leads to strain degredation? what is the purpose of cotony mycelia in natural settings? If mycelia was grown on compost,w ould it ever lead to cottony mycelia?


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2465058 - 03/23/04 09:33 PM (13 years, 3 months ago)

Cubensis mycelium has a tendency to be rhizomorphic when it is vigourous.  This is not absolutely truthful.  Some strains of cubensis are less rhizomorphic, and are still very vigorous.  Some strains seem to posses both rhizo and cottony sectors even when they are pure.

Starting with multispore on agar you may see some rhizo sectors.  You transfer to new plates, and the majority is rhizo, and some cottony.  You isolate only the rhizo, and transfer it to new plates.  This plate does NOT sector. It is P0, your first pure substrain(dikaryon).  This culture is replicated to 10 new plates.  Each of those is P1.  You store nine and use one to generate 10 new plates of P2.  You can continue this all the way out to P10 with cubensis, and experience NO SECTORING, on average.

Stametes recommends using cultures as close to P1 as possible for commercial grow operations with lots of money at stake.

A rhizomorphic culture(pure) might turn cottony based on substrate change, or other environmnetal tweaking.  It may also change to cottony based on mutations of a single cell, that then replicates into an entire sector, and then possible new plates that got made up from these bad sectors.  This is AGAR degeneration( visible on agar).

Stametes also recommends not doing more then 3 transfers from G1.  Inother words, you use a pure agar cultured strain(rhizo) to inoculate a jar of substrate. This is G1.  You use one jar of G1 to inoculate 10 of G2.  You use one the G2 to inoculate G3.  He recommends NOT transfering to G4, due to Contamination risk and possible Mutations.

Degenration is a strain that once fruited great at a certain environmnet Not producing up to par any more in that environment.  It can be expressed as slower colonization times, slower pinning times, fewer fruits, destorted fruits, sporelessness, and on AGAR it can be seen sometimes as a Pure rhizomorphic cutlute starting to sector away from pure rhizomorphic growth.  Linear types, and cottony types, and matted types might start sectoring out.  This usually means mutations have occured.  You either transfer only rhizo sectors of the plate, or you go back to one of your younger pure cultures for any future work.

PROBLEMS with storing cultures(STRAINS) is you end up with way to many, and they have to be revived at least every six months or so just to be safe.

IE.  Youy store nine P1 cultures, you want to revive at least 1 every six months and transfer to new plates.  You do the math, it EQUALS a SHIT LOAD OF PLATES per STRAIN(SUBSTRAIN).

Rhizo to cottony is usually a bad sign.  Most Cottony cultures wiull become rhizo in the casing, just not on agar.  RHIZOMORPHIC GROWTH in a casing is TYPICAL, even from substrains that are cottony on agar.

This RHIZO/COTTONY issue is helpful primarily in seperating vigourous growth on AGAR from multispored species LIKE CUBENSIS. But it is not absolute, becasue some STRAINs of cubensi are cottony by nature on Agar, but even these will express different forms of cottony mycelium and only experience with the Strain will assist you in determining which sectors are more vigorous.

Excuse the RUN ON explanation, but it happens. :tongue:


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Offlinestarspawn2
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2466501 - 03/24/04 05:42 AM (13 years, 3 months ago)

NO SECTORING after 9 generations?Thats amazing.I see sectoring all the time from mycelium cloned from a fruitbody.How many substrains on average grow from a mulitspore inoculation?


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: starspawn2]
    #2466650 - 03/24/04 08:38 AM (13 years, 3 months ago)

A lot. If you can see the spores when you put em on a plate you have way way to many.


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Offlineragadinks
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2466667 - 03/24/04 08:56 AM (13 years, 3 months ago)

Quote:

It may also change to cottony based on mutations of a single cell, that then replicates into an entire sector, and then possible new plates that got made up from these bad sectors. This is AGAR degeneration( visible on agar).



Does that mean that the mycelium also can "adapt" to the substrate ( for better or worse ) without a "sexual" reproduction stage in between ?


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OfflineAnnoA
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Re: Rhizomorphic and "cottony" mycelia [Re: ragadinks]
    #2467269 - 03/24/04 12:57 PM (13 years, 3 months ago)

It definitely does this, and this is the reason why one should always change agars so the mycelium doesn?t get too used to one nutrient formula.


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: ragadinks]
    #2476442 - 03/26/04 12:41 PM (13 years, 3 months ago)

The genes of a dikaryon are set. The interaction of the genes and the environmnet create the phenotype. Growing on a single agar medium repeatedly may cause that particular Dikaryon to become dependent on that particular formulation. Usually it is overcome by switching media formulations so that No enzymes are favored over others(all the genes responsible for producing the enzymes neccessary for eating substrate remain active).

Mutations can just be due to poor repllication of the nuclei, and lead to bad sectors arrising from this bad cell.

Certainly changing the media composition can change the appearance of the mycelium as well. But usually it is seen throughout the entire culture, not just as sectors. Culture A may look rhizo on one media formulation, but it may look linear on another.

Sexual reproduction(meiosis) envolves recombination of the genes. This means new types can appear, and old types can be reproduced.

Each new cell is arrising from a single cell. If that single cell is mutated, then all cells replicated from it will be mutated. This can cause sectoring.

A colony of cells can apppear one way on PDA, and that same colony can appear another way on MEA. The addition of Yeast, peptone, etc... can change the appearance even more. This is not mutation, but Phenotype expression of a genotype based on alteration of the environment(media formulation). The gentic code is the same, but the resultant appearance of that mycelium is different.

Pure cultures of a single Dikaryon. Multispored cultures could have many dikaryons present, and the sectoring can just be each colony working themselves out.

If you are working with Single Dikaryons, it is advised to rotate media formulations.


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Offlineragadinks
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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2476796 - 03/26/04 02:05 PM (13 years, 3 months ago)

thanks for the good explanation.
Is there a way to "revive" the encymatic capabilities of a phenotype that has become used to a certain agar medium over a prolonged period ?


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: ragadinks]
    #2484298 - 03/30/04 06:24 PM (13 years, 3 months ago)

Either go back to an older culture, or just put the culture on new substrate and let it try and work it out. After some lag time it should start growing again on the new media. If not, go back tto older isolate, or reisolate a new one from spores.


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Re: Rhizomorphic and "cottony" mycelia [Re: ]
    #2484703 - 03/30/04 08:19 PM (13 years, 3 months ago)

I was asking because I wonder if the mycelium looses it's capabilities of growing on another type of substrate at all or if these capabilities of producing the encymes for another kind of substrates just get dormant.
In the first case the mycelium would mutate and in the second it would just degenerate. Is that right ?


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Anonymous

Re: Rhizomorphic and "cottony" mycelia [Re: ragadinks]
    #2484776 - 03/30/04 08:37 PM (13 years, 3 months ago)

You are correct. Both cases are possible, dormancy is more likely. If you did get a mutation, then all mycelium produced from that sector would be incapable of digesting anything but similar substrate.

Most often, you just experience a lag period when transferring to new substrates. Similar to that you can experience when spawning, just longer.

Most of this is just for knowledge sake, as it is highly unlikely for cubensis to get substrate specific, it eats such a broad range of food.

It is far more likely with other types of fungi, and specifically with PAthogenic fungi. These are typically mutations within the pathogenic fungi. Severe enough that the new types will not mate with the old types. SPECIATION.

At best you might experience some lag time, and some reduction in vigor on off types of substrate with cubensis. I don't think you can acheive total substrate specificity without a mutation occuring. At least a specificity that is passed on too offspring.

I suggest you stick with waht works for you, and if you see some changes occuring in the quality of a clone. ISOLATE a NEW ONE. The whole purpose of cloning is to have a guranteed producer in your cultural specs. If the clone stops producing in those specs, it is worthless to you.

I isolate clones that perform well throughout the whole lifecycle. MEA>Rye>Manure. That clone might not perform the same if I go PDA>Millet>Straw. So varying the substrate neutralizes the clones benefit if substrate specificity was a selecting factor.

If I am selecting for a clone that produces on any substrate within a range of choices, that performs well in specific humidity/temperatures, then varying substrate is neccessary, while maintaining my chosen environmnetal specs for everything else.

this latter situation is probably the better way to go.

To me the best selection factor is Yield on a particualr end substrate, regardless of AGAR/SPAWN composition. So varying the agar formula, and spawn formula can only help in maintaing vigor of my clone.

Hope that helps.


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Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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