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InvisibleYidakiMan
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agar isolation
    #1882713 - 09/04/03 09:59 PM (13 years, 11 months ago)

I am about to go through the process of isolating a shiitake strain. I am curious to understand how many isolates one must work through to find what they are looking for. As an example, in GGMM Stamets claims that his strain of shiitake, CS-2, voluntarily forms primordia on MEA in three weeks and can fruit as quickly as 14 days from inoculation onto supplemented sawdust. I have also read that many farmers use non-sporulating strains of oyster. How many isolates did he work through in order to find this "unique" strain?


Edited by YidakiMan (09/04/03 10:03 PM)


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Offliner05c03
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Re: agar isolation [Re: YidakiMan]
    #1882900 - 09/04/03 10:49 PM (13 years, 11 months ago)

Realistically you are talking about lots of time and money to find strains that you want. At this point you probably only need something that fruits which you should be able to get fairly easily, especially with oysters. If you are thinking that you are going to go immediately into production then I would save yourself some time and money and purchase a well characterized strain.

To characterize strains you need standardized media, and consitent fruiting conditions to be able to draw solid comparrisons. Hours and hours and dollar and dollar can go into characterizing strains. You may want to familiarize yourself with simply getting something fruit rather than characerize strains. Take it step by step and be patient. Learn and apply.


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InvisibleYidakiMan
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Re: agar isolation [Re: r05c03]
    #1883582 - 09/05/03 01:52 AM (13 years, 11 months ago)

I was pretty sure that finding strains that fruit incredibly fast or non-sporulating would require a huge amount of isolations to pick through. I was simply going to find a nicely fruiting strain. Thanks for your insight.


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Offliner05c03
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Re: agar isolation [Re: YidakiMan]
    #1886013 - 09/05/03 06:32 PM (13 years, 11 months ago)

Oh, that was not at all clear from the manner in which you asked your question. Finding something that fruits should not be that hard. Happy growing.


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InvisibleYidakiMan
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Re: agar isolation [Re: r05c03]
    #1886367 - 09/05/03 08:27 PM (13 years, 11 months ago)

To put it more clearly; I will find something that fruits, I want to find something that fruits in 14 days. =)


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Offlinepluteus
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Re: agar isolation [Re: YidakiMan]
    #1906811 - 09/11/03 08:18 PM (13 years, 11 months ago)

If you want maximum control and influence over your strain breeding trials, above and beyond what most amateur cultivators attempt, it is useful to be able to carry out isolations of cultures derived from single spores. Monosporous cultures (which are usually monokaryons) can then be mated in selected combinations and the quality of cultures from successful matings assessed. In primarily or secondarily homothallic species (e.g. Agarics bisporus), monosporous cultures will fruit without mating. Although this is a more straightforward system for choosing desirable strains, it presents the breeder with less opportunities for changing genetic properties. Saying this, compelling some homothallic species to mate can be done.

How to isolate single spore cultures? One method involves water dilution of spore mass to a factor where a few drops of water will contain only a few spores. After placing a few of these drops onto an agar plate, emerging cultures can be verified as monosporous by using a microscope. A 'spore cutter' device can then be used to separate the emerging cultures into their own dishes. More complicated methods involve the isolation and manipulation of individual spore tetrads.

Incidentally I have never heard of these techniques being applied to P. cubensis. They would be critical in attempts to mate different strains.


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InvisibleYidakiMan
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Re: agar isolation [Re: pluteus]
    #1907283 - 09/11/03 10:23 PM (13 years, 11 months ago)

Very interesting. Thanks for the info pluteus.


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OfflineMycena
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Re: agar isolation [Re: YidakiMan]
    #1908734 - 09/12/03 07:33 AM (13 years, 11 months ago)

Buy a FP block and clone it?

I think all this talk of senescence is BS and designed to put people off cloning shop bought mushrooms and extend protection to those in the spawn industry
Ive never had a species go senescent except as the direct result of giving it crappy media for a long long time.
Keep on shifting the nutrition and theyre fine
What i think is more of an issue is clonal dgeradation via mutation and selection of poor fruiting muatnts from within the cell line

In effect each time you take a small sample and subculture it youre taking a subset of the cell population and allowing it to grow out- mutants always accumulate through natural and chemical exposure but on agar there is no selection filter.
eg a mutant may be a poor fruiter but if your not fruiting it youll never know and just keep on subbing it. In vitro the cell pouplation is being actively selected for life in vitro. The cells are being chalenged to grow on a poor homogenous media in a sterile environment so the cell lines that cope best in this situation are growing in proprtion to the original colony.
If you were to fruit this colony you would seriously put pressure on those 'lab animal' cell lines and push the advantage back to the hardy cell lines - and if you then clone the best looking fruit bodies youll see the strain is reinvigorated.

Evidence to support my hypothesis is all about us in nature.
many woodloving and grassland Mushrooms species persist almost indefinitely.
SRA, Agaricus, and most famously Armillaria.
These species move through the environment clonally as well as by spores but particularly in the case of several Armillaria species innoculation by spores i very rare - many fruitbodies are even sterile.
These persist for many years, and in the case of Armillaria even THOUSANDS of years.
The difference is that in the wild the cell ines in each colony are subject to contsnat selection pressure to stay healthy. They are constantly challenged metabolically have access to complex nutrition.

I sincerely think this simple age related theory in the senescence problem will be proven in future to be a fallacy .


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Re: agar isolation [Re: Mycena]
    #1908748 - 09/12/03 08:06 AM (13 years, 11 months ago)

In our lab we use a microscope (a specialized one with the objective lens coming underneath the sample rather than from the top). We sepearate single conidia. with glass tools that we make from pipettes.

As far as senescence, I think you are right to a certain extent it is not so much age but the what they are fed as they actively grow. The more you subclone, the more chance for the accumulation of "bed" cell lines. It is very notable in the plant pathogenic fungi with which I work. However, some fungi, probably not most of the one we like to grow are very dependent on storage conditions as well. It seems to be the specialized qualities such at the ability infect this or that plant that go first, presumably those abilities are under the control of fewer or single genes which and are therefore easier to mess up with slight changes to the DNA or to expressions systems. In nature clonal cells that pop up are quickly killed, thus the prolonged clonal exisitence of the fungi.


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OfflineMycena
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Re: agar isolation [Re: r05c03]
    #1908808 - 09/12/03 08:52 AM (13 years, 11 months ago)

yes i am very afamilar with what you are saying about species whose existence depends on interaction with another species
ie Mycorhiza and or pathogenic species
It seems do lose their ability very quickly to reform relationships

Though i dont think this phenomenon is the same as the senescence that saprophytes undergo.
In some cases it may be media related - the right media just hasnt been found to preserve the integrity of the culture, in other cases the evolved characteristics may be easily lost with the culture reverting to the more primitive lone existence strategy.
The lack of stimulation may also be a problem at the genetic level - complex systems interact to form mycorrhizas.
Using Rhizoctonia as an example - to some grasses and solanaceous plants it is a damaging pathogen whilst to many terrestrial orchids it is a mycorrhizal symbiont. The difference is in the communication between plant and fungus, if the plant can signal the fungus in the right way it can be harnessed to mutual benefit. If not it comes aboard anyway and is a disease causing organism
Perhaps this is also a media problem. High levels of sugars in the media and teh absence of complex organics and minerals may switch off genes expressing for the right RNA/ enyzymes need to form the connection - as a kind of complex form of catabolite repression
Its too easy to blame the fungus when in reality its our own inadequacies in formulating the correct media. Maybe even the lost isolates could be coaxed back to health if we could put them on the right media and then challenge them with suscptible plant hosts. the genes are all still there they just have to be provoked into expression


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Re: agar isolation [Re: Mycena]
    #1908854 - 09/12/03 09:41 AM (13 years, 11 months ago)

Hmmm, the pathogen rhizoctonia (Rhizoctonia solani, Rhizoctonia phaeseolus)is a pretty basic pathogen, nothing too special about the manner in which it infects plants, no gene for gene interaction like you see in powdery mildew or rust for example. Is this the same rhizoc that forms mychorrhiza with orchids?


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Offlinepluteus
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Re: agar isolation [Re: r05c03]
    #1908949 - 09/12/03 10:52 AM (13 years, 11 months ago)

Senescense is not just an evil conspiracy propogated by the mushroom cultivation industry, although it may be much more of an issue for some species than for others.

I think its likely there is wide variation in species' ability to grow indefinitely. Different species will have been under very different selection pressures for persistence traits. Species colonizing ephemeral substrates (e.g. obligate saprotrophs of sheep dung), which fruit very quickly, may have evolved less efficient mechanisms to maintain genetic stability in a single vegetative phase, whereas species with a constant supply of substrate can afford to invest in such mechanisms.

A related point. In the very long lived colonies of Armillaria spp., Marasmius oreades etc., it seems likely that the colony is repeatedly fusing with hyphae from its own and others' newly germinated spores. Even in a 'stable' dikaryon, genomic takeover of the mycelium by some novel nuclear types is possible. The high speed of nuclear migration would allow takeover of a colony even a few acres in size to occur within a year. Thus what appreas to be an ancient clone has staved off senescence by being open to nuclear replacement. (if this is true, then mitochondrial genes might be better than nuclear, although still problematic, if you were attempting to date the age of the colony from samples taken around its perimeter)

If this all sounds too farfetched for you then consider the simpler scenario of a 'bad' cell line arising within a very large colony. There would be a vast excess stable nuclear types available to selectively edge out the unfavourable lines. Although the colony would still experience differential, perhaps zoned genetic change, this would largely be in the form of neutral or nearly-neutral mutations, and so not such an impediment to indefinite, fertile, coordinated growth.

I doubt that these size-reliant control systems over genetic stability are as effective at the level of petri dishes, spawn bags, etc. Which explains why you observe many ancient colonies in nature, but artificial ones require much selective interference to persist, and can eventually fail.



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OfflineMycena
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Re: agar isolation [Re: pluteus]
    #1909120 - 09/12/03 12:07 PM (13 years, 11 months ago)

OK

rhizoctonia -
i just typed in rhizoctonia and orchid to google and voila
http://www.google.com.au/search?q=Rhizoctonia+orchids&ie=UTF-8&oe=UTF-8&hl=en&meta=

i already knew about this one cos at my old universit they are investigating the genes that make the difference between pathogen and ally because it aversely affects so many commercial crops and yet is essential for our native woodland orchids.At least some use R solani - and all around the world they use other species as well

I know in its usual infective state its not very specialised and for this reason its iteresting that it can also form such specific mycorrhizas.

Re: Pluteus
I think its likely there is wide variation in species' ability to grow indefinitely.

definitely - as you say its easy to begin to sort those who employ the perrennial strategy to the ephemeral - what are those again? theres a name pop. biologists give to these 2 startegies? r and K ?
anyway yes a Pan cyan or Poronia with a lifecycle covering a matter of weeks may be a lot harder to keep healthy in vitro than a long lived species due to less well developed genetic repair mechanisms
however i dont think it is a given that senescence will occur if the selecion processes are against it.
Commercial stocks of biosynthetic microbes senesce with repeated subculture but hand in hand with that commercial reality is the process in the other direction that created the super strains in the firtst place from less vigorous lower yielding parents - and the process can go weither way depending on the level of selection/pressure. Senescence is inevitable in the sense that in the end every lineage dies out - its a statistical inevitability that any one line will accumulate fatal flaws, but not before it spawns new and divergent daughter cell lines

Senescence and its inevitability seem almost like dogma in the OMC. I brought this up because people believe in things like P values and such without questioning their value. And Vendors charge much much more for 'young' cultures that are supposedly superior than cloned shop bought mushrooms on the assertion that age is somehow a ticking clock.
For a very small subset of organisms or cell types this is true (Some Bamboos, Endothelial cells). It was once believed that we had inbuilt biological clocks and our cells could only replicate so many times, the clocks (telomeres) have since been shown to be independant of ageing and senescence in mammals (and they are elongated routinely) so it brings into doubt the entire notion that strain age is an important factor

In order to accept this i need to see lots of supporting research, i want the mechanisms fully outlined and i want to actually see a strain senesce - so far no joy :frown:

In stamets GGMM it said eucalypts were unsuitable as a wood source for mushrooms. This was based on anecdotal evidence and now has been shown to be far far from the truth. I believed it and propagated the dogma till shown otherwise. So after that im very very sceptical about these apparent 'concepts' until they are fully explored incl. for whose interest the proposed rule works 



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InvisibleYidakiMan
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Re: agar isolation [Re: Mycena]
    #1910377 - 09/12/03 06:05 PM (13 years, 11 months ago)

I think what I am going to do is clone some specimens from the grocery store, fruit, print and isolate.


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Offlinepluteus
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Re: agar isolation [Re: YidakiMan]
    #1919205 - 09/15/03 08:38 PM (13 years, 10 months ago)

Mycena -
I think you do have good grounds for a gripe, and this is an important issue. Apart from a few commercial species, mushroom cultivation remains more 'art' than science, as it relies on techniques developed by a handful of 'visionaries' whose writings drift down to us underlings from the higher realms. Yes, these visionaries are excellent people, highly scientifically trained and all that, but surely it would be a better situation if cultivation had been a major concern of the international scientific community from the word go? It is, however, easy to see why it was not, and hence why we now suffer from this historical lack of data.

Encouragingly, however, if you scan the current literature you will find that many more species have started to be used in research that addresses questions related to cultivation, including senscence. Of course there is always good old Agaricus bisporus for which there is abundant data about how fruitbody productivity, abberancy, susceptibility to disease, etc., change with the age of a culture and its total extent of growth (and much of this research has not been concocted or funded by growing companies).

You say, "It was once believed that we had inbuilt biological clocks and our cells could only replicate so many times"
This is still a valid belief. Loss of telomere length is only one of a great number of mechanisms implicated in cell ageing. Also, there are numerous examples of cell types in the human body which are strictly limited in the number of times they can divide (e.g. the cells that produce eggs, certain types of neurones, the cells in the lens of your eye). Even considering that these are consequences of inbuilt developmental restrictions, and not a result of senescence, I still do not agree with your statement that it is a "very small subset of organisms or cell lines" that are, in their natural state, constrained in their ability to persist. Were this true than the majority of organisms would have indefinite lifetimes, and the ages at death of individuals in a species would show a very different distribution from what is observed, with some being extraordinarily long-lived.
I think you are right though, to believe that fungi are so different in their biology that no a priori assumptions about the probability of their senescence should be made. I will look out for specific research into this issue.






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