Home » Biology, Evolution, Science » Gene Induction in Fungi – Lamarckian?

Gene Induction in Fungi – Lamarckian?

As some of you may recall I wrote that I was experimenting with laboratory propagation of volvariella volvacea (Chinese Straw Mushrooms). Recently, among several other lines of R&D, I was experimenting with hydrogels as a nutrient media. So far I’ve been using them as an agar replacement with mixed results. I think the mixed results are due to uneven moisture distribution in the fine powder form I was using but that’s neither here nor there. Since the hydrogels can be loaded with nutrients at room temperature (the big advantage over agar) I decided to play around with another sterilant that would decompose at temperatures required to melt agar. I’ve been extremely successful using ampicillin at 1mg/20ml to prevent bacterial contamination in agar cultures – haven’t had a single bacterial infection in hundreds of agar plates. Ampicillin however breaks down quickly at temperatures over 60C so it must be added to agar at a critical stage after it’s cooled down (agar melts at 95C) some but before it solidifies (about 40C). This requires pouring fast and keeping a 60C water bath on the bench. However, ampicillin is so inexpensive it can be considered free of cost compared to wide spectrum antibiotics that survive pasteurization and autoclave temperatures. Once poured, ampicillin plates must be refrigerated until use as ampicillin in solution breaks down quickly at room temperature (a matter of days).

Anyhow, the new sterilant I was experimenting with is hydrogen peroxide (H2O2). I added a 3% (drugstore concentration) h2o2 solution to liquid nutrient at various ratios from 0.5ml/100ml up to 5ml/100ml, soaked a bit of hydrogel with the solution in culture plates at room temperature, then innoculated them from a vigorous volvariella colony. I did this all in the open air taking no particular sterile precautions (I usually use disinfectant, rubber gloves, scalpel sterized in alcohol flame, and a HEPA laminar flow hood to conduct sterile transfers) doing it with just an alcohol swab on the scalpel and syringe needles and on my kitchen countertop next to the toaster & blender – clean but definitely nowhere near sterile. The lower h2o2 concentrations allowed vigorous if somewhat reduced growth but about half of them contaminated with penicillium fungus (all the plates contained ampicillin so only fungal contamination was possible) after a few days. At higher concentrations of h2o2 new growth was completely arrested but none contaminated. After about 2 weeks I was ready to draw conclusions then move on.

Then a vicious series of thunderstorms with 60 mph winds rolled through the area and I had to stay on my boat for four days riding them out and making sure it remained securely anchored. When I returned home and checked my incubation chamber I was in for a big surprise. One of the h2o2 cultures (the highest concentration I tried at 5/100) that had been completely arrested for the prior two weeks, had bloomed and taken over the entire plate with an aggressive healthy volvariella colony. The rest were unchanged. Just one took off and when it took off it took off like there was no peroxide.

Curious, I wrote to Dr. Rush Wayne who’s pioneered the use of h2o2 in mushroom culture and whose procedures I purchased for a guideline (he’s never worked with this species) and asked if he’d seen anything like this. He said he hadn’t experimented with concentrations that high but he said it would be what he would expect if volvariella had used gene induction to turn on an h2o2 decomposing enzyme. H202 is a naturally occurring sterilant present in the environment and most living things produce enzymes that decompose it. Those enzymes are what cause the fizzing when you pour it on an open wound. Spores don’t have enough of the enzyme present to survive germination in a low h2o2 concentration but a healthy fungal colony already does and hence its efficacy as a sterilant in mushroom culture. One merely has to insure that the nutrient substrate doesn’t have h2o2 decomposing enzymes in it. Almost all processed nutrients have had those enzymes destroyed by the heat of pasteurization which includes the potato/dextrose broth I was using.

Dr. Wayne said some organisms may have latent genes for enzymes that can decompose h2o2 much more rapidly than normal and can turn on those genes through “gene induction” if they encounter high concentrations. None of the mushrooms he’d experimented with had exhibited gene induction for h202 decomposing enzymes. But volvariella is an odd duck amongst mushrooms in its voracious apetite over a wide range of substrates, inability to survive temperatures below 45F, dying (or at least going dormant with asexually produced chlamydospores if conditions are right) easily when it runs out of food (which drastically limits shelf life of harvested mushrooms), and senescence when recultured for very many generations (it mutates rapidly when stressed like when it runs out of fresh agar surface to colonize).

So anyhow, I now have a volvariella colony that mutated beneficially within a matter of weeks in a most Lamarckian way in response to environmental stress. Its progeny inherit the ability to tolerate high h202 concentrations and show no loss of fitness in the absence of h202 (I recultured the colony back onto h202-free agar and it looks just like the control colony recultured at the same time). We’ve blogged here before about Scripps discovery that e.coli can rapidly adapt to antibiotics by enabling certain genes to mutate at extremely high rates and that the antibiotic resistance is heritable (and horizontally transferrable through plasmids). I speculated from that that eukaryotes probably had the same capacity to adapt since they ostensibly evolved from prokaryotes. Volvariella is a eukaryote and I just witnessed it evolving faster than random mutation and natural selection could possibly account for. Yet another nail in the RM+NS coffin…

  • Delicious
  • Facebook
  • Reddit
  • StumbleUpon
  • Twitter
  • RSS Feed

25 Responses to Gene Induction in Fungi – Lamarckian?

  1. DS,

    Great sporesmanship!

    :lol: -ds

  2. Hey Dave,

    You said that it was about two weeks later that you saw growth. I don’t know anything about these mushrooms but I’m assuming you aren’t growing them anaerobically. Unrefrigerated hydrogen peroxide will most likely convert to water within that timeframe in the presence of oxygen. If I were you I’d replate the resestent colonies on plates with more H2O2 and see if they are still resistent. The more likely explanation for your phenomenon is that some of the colonies (spores?) managed to survive long enough until the H2O2 had been naturally depleted.

    When hydrogen peroxide decomposes it produces 02 and water. If it didn’t store well at room temperature in the presence of oxygen it wouldn’t last long in the bottles at the drugstore because its decomposition would just increase the 02 content in the sealed bottle and increase the gas pressure. In fact it stores quite well at room temperature (years) in the presence of oxygen without decomposing and gas pressure doesn’t build up in the bottle.

    Actually the cultures are WERE grown to that point in a gas-tight environment. They have enough oxygen to grow across the plate then I switch to a lid with a microporous filter for longer term storage, periodically opening the filter so gas can be exchanged if they aren’t refrigerated to arrest their growth. If I let the gas exchange go constantly the little buggers will reduce a 1/4″ layer of agar into a dried out paper thin disk in a few weeks. For really long term storage I incubate about 2 weeks at 87F until they get a nice orange tint which indicates they’ve formed asexual chlamydospores then I seal them up gas tight so they don’t dry out and put them into a refrigerator modified to maintain 50F. They take about 5 days to recover (chlamydospore germination) after coming out of refridgeration. This species is notoriously difficult to maintain in culture. Every other mushroom culture can be stored for years at 34F (normal refrigeration temperature) but with these that temperature will kill them and the chlamydospores. Chlamydospores aren’t true spores but rather a few cells twined into a ball with thickened cell walls to inhibit moisture loss and survive dry (but not cold) periods. I recently acquired a nice biological light microscope (Swift M1000 series) to investigate the chlamydospore formation. I’d want to experiment to find a way to collect chlamydospores for long term dry storage and transportation at room temperature. The problem with them in a live culture is that unless the agar is totally dried out they resume growth after a several days at room temperature and have a limited shelf life. They’re really aggressive and can extend mycelial filaments about a half inch through open air looking for food and if they have a glass wall to climb will go several inches along the glass looking for more food. I found half-pint wide-mouth mason jars to be the most convenient culture vessel with standard gas-tight metal lids and/or plastic lids with microporous filters depending on the usage. In less than a week a freshly innoculated jar with a 1/4″ of agar on the bottom will become so thick with snow white mycelia you can hardly see inside the jar. Sometimes a particularly strong colony will even cover the inside lid of the jar. Controlling the growth rate without killing them is tricky. -ds

  3. Barry Hall actually pinpointed specific insertion sequences that this happened for in E. Coli:

    http://www.ncbi.nlm.nih.gov/en.....query_hl=1
    (summarized here: http://baraminology.blogspot.c.....istic.html )

    As for the quick mutability of E. Coli under pressure, there is some evidence showing the specific non-random mechanism that E. Coli uses, and this can actually be used to predict where the sequence changes will occur:

    http://jb.asm.org/cgi/content/full/182/11/2993

    (summary along with a few other papers here: http://baraminology.blogspot.c.....nesis.html )

    From the paper:

    “What is the probability that these structures actually exist in vivo and constitute precursors of background mutations in nature? One kind of evidence for their existence is the striking correlation of deletion end points with tandem repeats and the ends of potential secondary structures. The leuB, argH, and pyrD mutations in E. coli all occur at the end of predicted stem-loops (111). The lacI gene has frequently been used as a model system for investigating these correlations by comparing its nucleotide sequence (26) to those of various mutant strains. Among the sequenced deletion mutations in lacI, 7 occur in tandem repeats, 6 consist of deleted stem-loop structures, and 4 of these 13 mutations share both characteristics. Although three remaining mutations fail to coincide exactly with predicted vulnerable sites, they may be explained by different DNA secondary structural intermediates including interstrand misalignments (26, 88, 92). Todd and Glickman (99) analyzed 102 amber and 71 ochre mutants in the lacI gene, correlating mutation hot spots with the locations of predicted unpaired sites, many of which are located in stem-loop structures. Mutational hot spots are highly localized, and 50% of the nonsense mutations arose in a segment comprising only 6% of the DNA sequence analyzed. Each hot spot is found to be located at an unpaired site within potential secondary structures. Clearly, these correlations depict causal”

  4. Err, you have heard about gene regulation, haven’t you?

    Bob

    Sure, but the hypothetical peroxidase gene is turned on in the daughter cells without them needing weeks to adapt. A probable mechanism is that a regulatory protein is constantly produced in very small quantities. Just a small quantitiy is enough to bind to a locus near the peroxidase gene and turn it off. Thus a very small signal controls a very large signal. Little cellular energy is wasted in the normal case of not needing large quantities of peroxidase by only needing tiny amounts of inhibitor. In analog electronics this is called signal amplification. It works the same way in a cell. Now lets say the inhibitor is destroyed by peroxide so that during mitosis there is no inhibitor to bind to the newly created daughter DNA. The daughter is thus born with the peroxidase gene uninhibited and she’s immune to peroxide’s effects at the cost of however much cellular resources (energy & raw materials) it takes to produce peroxidase. Presumably the inhibitor keeps on being produced in small quantities and removal of the peroxide will cause peroxidase production to cease. But that’s still evolution. Darwin’s Finch beaks return to normal size when drought conditions are removed and that’s still called evolution. This is Lamarckian inheritance of acquired characters and it certainly isn’t driven by random evolution and natural selection – it’s an adaptive, heritable response to a specific environmental change. Just because it’s epigenetic doesn’t mean it isn’t Lamarckian inheritance. Lamarck didn’t know know DNA from donuts. -ds

  5. There is an interesting paper here:

    http://www.rae.org/introns.html

    “The author concluded that the new knowledge related to introns supports the intelligent design worldview.”

    Perhaps more relevant to this blog are papers like:

    http://www.ncbi.nlm.nih.gov/en.....t=Abstract

    which investigate the loss and gain (conservation) of introns (information)in fungi.

    Experiments involving conservation of intron information could be a rich source of empirical evidence supporting and furthering theory’s like Dr. Demski’s Conservation of Information.

    IDers sucessfully take pot-shots at targets like RM+NS. Offering real evidence of advancement in the very promising new fields of research would be a more decisive indictment of neo-darwinism

  6. The paper by Barbara Wright is interesting. When microorganisms are starved for an amino acid, they derepress enzymes able to make at least some of the amino acid. So it is the derepressed biosynthetic genes that are now madly transcribing and therefore mutating, in the hope of improving their ability to make more of that essential amino acid, perhaps by a mutation converting another closely related amino acid (in excess) to the scarce amino acid. It’s another example of a feedback in Biology.

    A cell expressing a normally repressed gene in response to need is de rigueur – that’s what cells do. However, when a cell reproduces and the daughter cell comes out the gate with the gene already expressed, that’s a different story. That’s what happens to stem cells. A stem cell only has to be cued to turn into a muscle cell one time then all the cell’s descendents automatically become muscle cells and the process is difficult if not impossible under any natural circumstances to reverse. This is what may have happened to the H2O2 tolerant colony. Or it may be a temporary adaptation. Mushroom mycelia are very good at optimizing enzyme production for specific food sources if you give them time to adjust and in the absence of that food source they cease being optimized for it after a while. This very well what may be happening with the h202 tolerance although that doesn’t mean it isn’t Larmarckian. It only means it’s epigenetic and temporary rather than a permanent genetic modification. If I can make another electronics analogy there might be something acting like a flip-flop – i.e. once a gene is expressed it remains expressed as long as its own protein product is present. The thing that turns it back off (resets the circuit) is a different input than what turned it on. That would mean the protein product just being present in the cytoplasm of a cell undergoing mitosis would automatically have the daughter cell expressing the protein as soon as she’s born and possibly, as in the case of differentiated stem cells, there’s nothing natural that resets it back to the original state. -ds

  7. Conservation of (intron) Informaton experiments

    http://mweir.web.wesleyan.edu/.....theory.htm

  8. When hydrogen peroxide decomposes it produces 02 and water. If it didn’t store well at room temperature in the presence of oxygen it wouldn’t last long in the bottles at the drugstore because its decomposition would just increase the 02 content in the sealed bottle and increase the gas pressure.

    True. It’s fairly stable without a catalyst and is usually ok sitting around in that brown plastic bottle, although they usually pack it with a stabilizer. But there’s a whole host of organic molecules that could rapidly increase the decomposition, as well as metals and small inorganic molecules. And if it’s in a basic environment, decomposition will also go faster. I’d be curious to see what would happen if you tried to grow the survivors on H2O2 rich media. I’m all for resurrecting Lamarck because I think epigentics is often overlooked, but I think we should wait a bit before we start the rituals. Peroxides are generally stable but one good free radical and they come crashing down like a house of cards (well, maybe that’s a bit exagerated but….)

    Sure I’ll test some more. This time I’m going to use real agar instead of hydrogel to make sure there’s just a single variable and in the meantime I’ve got it recultured on h2o2 free agar. A few other factors are indicative it wasn’t h2o2 decomposition. First, it was the only long term survivor above a concentration of 2 parts in 100 which is a little puzzling but contrary to decomposition or the others should’ve decomposed ahead of the one with the highest concentration – this makes me wonder if it wasn’t a genuine mutation instead of a gene induction. Second, the nutrient solution I used was autoclaved which destroys any and all natural peroxidases. Third, Dr. Wayne’s years of research has not revealed any inefficacy after just 2 weeks even at 1/10th the concentration I used. He stores uninoculated plates at room temperature with no noted time restriction. He specifically says room temp storage of uninculated plates is a benefit of peroxide as storage in a fridge causes condensation which increases contamination risk. Fourth, I have a sensitive electronic pH meter, recently calibrated, and the nutrient solution is slightly acid at 6.7 – my tap water reads alkaline at 9.0 but has practically zero buffer capacity so it turns acid at the drop of a hat (or a drop of potato broth in this case) and the hydrogel is completely inert. Fifth, there should have been contamination in some of the lower concentration duds as I opened up the cultures for 10 seconds and literally breathed on them at least two or three times but there was no contamination except at the lowest concentrations and only half of those contaminated. Sixth, when growth did resume it did so overnight and returned to what appeared to be normal (completely taking over a plate in 3-4 days). If it was because of decomposition I would expect a gradual restoration of growth not on/off like a light switch. So the only thing I’m puzzled about is that only one culture with high h2o2 concentrations survived and prospered and it happened to be the highest one. -ds

  9. DS: I take it you’rw now admitting that this was wrong:

    So anyhow, I now have a volvariella colony that mutated beneficially within a matter of weeks in a most Lamarckian way in response to environmental stress. I

    (emphasis mine)

    and that it’s just simple gene regulation. Incidentally, how do you know that the new mushrooms were produced by spores, and not through hyphal growth?

    Bob

    Where did I say anything about any fruiting bodies? These are mycelial cultures. I’m admitting nothing. Descent with modification took place. New cells are created with high h202 tolerance that the parent cells didn’t have. Whether that’s “mutation” depends on how you define mutation. I said the change (techincally that what’s mutation means) is probably epigenetic. Does epigenetic change suddenly not count as evolution because you don’t want it to? -ds

  10. The peanut gallery represented here by Bob OH appears to be not able to discriminate between gene regulation in a single cell and a change in how a gene is regulated in daughter cells. If a single cell produces an enzyme in response to 14 days of exposure to a stimulus that’s gene regulation. If her daughter cells are created with the enzyme already in production, i.e. no need for the long exposure to the stimulus, that’s descent with modification. It may not be a change in DNA that caused the modification in the daughter. Perhaps Bob and company don’t know about epigenetic inheritance whereas I do.

    Heritable changes in gene function without DNA change

    This includes the study of how environmental factors affecting a parent can result in changes in the way genes are expressed in the offspring [1].

    1. R.A. Waterland, R.L. Jirtle, “Transposable elements: Targets for early nutritional effects on epigenetic gene regulation”, Molecular and Cellular Biology 2003 August 1;23(15):5293-5300.

    I surmised a mechanism something like this:

    Chromatin-marking systems. Proteins or chemical groups that are attached to DNA and modify its activity are called chromatin marks. These marks are copied with the DNA. For example, several cytosines in eukaryotic DNA are methylated (5-methylcytosine). The number and pattern of such methylated cytosines influences the functional state of the gene: low levels of methylation correspond to high potential activity while high levels correspond to low activity. While there are random changes in the methylation pattern, there are also very specific ones, induced by environmental factors. After DNA replication, maintenance DNA methyltransferase make sure the methylation pattern of the parental DNA is copied to the daughter strand.

    Bob, I think you need to take some advanced biology classes if you don’t know about epigenetic inheritance. You also need to get a few more clues on the life cycle of perfect fungi (mushrooms) if you don’t know that mycelial colonies grow by asexual budding like imperfect fungi (yeast, for example) and really only differentiate themselves from imperfect fungi by the production of fruiting bodies (the mushroom proper) that have sexually produced spores. When a cell reproduces by budding it produces a clone of itself. You probably know that. When the daughter cell inherits an acquired change that’s Larmarckian evolution in action. Thanks for playing. -ds

  11. Where did I say anything about any fruiting bodies? These are mycelial cultures. I’m admitting nothing. Descent with modification took place.

    If the cultures are mycelial, then they are the same individual. Hence, no descent.

    I was asking about whether they were produced from spores because I do know something about the life cycle of fungi:
    I spent 5 and a half years doing research in mycology, and my PhD is in fungal plant pathology. The point with mushrooms is that if they are only hyphal, then they are the same indidivual: two mushrooms produced by one hyphal culture are parts of the same individual in the same way that my left arm is a part of the same individual as my right arm is. Would you claim that your liver cells (say) have “inherited” their function when they were produced? That’s not evolution: it’s development.

    Epigenetic inheritance isn’t an issue, because inheritance isn’t an issue.

    Bob
    P.S. I was hoping to be banned with a bit more of a flourish than “Thanks for playing.”. I’m really disappointed. :-(

    You should ask for a refund on your PhD, Bob. Mycelia are colonies of discrete individual organisms. Any individual cell in the colony can and will produce a new colony. They are composed of individual cells that reproduce by budding, also known as vegetative growth. They may or may not all be clones. If they are all clones they are what’s called a pure strain or monospore culture. I’m working with a pure strain. All the individuals are descended from a single spore. Pure strains are used in commercial mushroom production because they exhibit consistent, predictable performance (senescence issues aside). But here’s what you seem to be missing, Bob. Descent with modification doesn’t stop just because I’m working with a pure strain that is reproducing asexually. Individual cells can and do continue to descend with modification in mycelial colonies. That’s how evolution works, Bob. Daughters aren’t always perfect copies of their mothers. Colonies can also be multi-spore or dikaryotic where the individuals in the colony are descended from more than one spore and this is typical in nature where reproduction from season to season is accomplished by the haphazard germination of multiple sexually produced spores. In multispore colonies it’s a crapshoot as to fruiting capacity, optimal substrates, etc. which is why in commercial production we use monospore cultures as long as we can maintain the the pure culture without it becoming senescent. I can spoonfeed this stuff to you if you stop making faces and spitting it out. -ds

  12. Hark! What’s that sound I hear from the peanut gallery? I do believe it’s the sound of crickets chirping! :cool:

    You’re not banned Bob, in case you thought that. Feel free to keep on playing if you think you have a move left.

  13. ‘Inoculated’ has only one ‘n’, if you remember, Dave.

    Curses. I’ll will console myself with the thought that even a blind squirrel finds an occasional acorn. -ds

  14. First of all, epigenetic and development are different sides of the same coin. Whatever the cause of this observation, how is this a nail in the coffin for RM+NS ? It is certainly true that recent discoveries in epigenetics have given Lamarck some new relevance and DNA is certainly not the only molecul relevant to evolution and inheritance – I mean, we still have to inherit the endoplasmic reticulum.

    But disregarding the semantic argument here, how is this observation a nail in the coffin for RM+NS ? In the sense that RM+NS can’t explain EVERYTHING ? Or in the sense that RM+NS hardly explain ANYTHING ? And how does our new understanding of epigenetics/development suggest a designer ? Does it have relevance to a design inference ? How so ?

    In the sense that RM+NS can’t explain EVERYTHING ?

    Everything? I’m still looking for the first thing it can explain other than death of the individual and extinction of the species. -ds

  15. Further spoonfeeding of Doctor Bob.

    http://www.genetics.org/cgi/content/full/164/2/479

    Genetics, Vol. 164, 479-485, June 2003, Copyright © 2003

    Sex Slows Down the Accumulation of Deleterious Mutations in the Homothallic Fungus Aspergillus nidulans

    A good example is the fungus Aspergillus nidulans (Fig 1). In its vegetative state, it consists of a mycelial colony that typically originates from a single haploid spore. All nuclei in this colony are therefore genetically identical, except for novel mutations that have arisen spontaneously during the growth of the mycelium.

    And of course for heritable epigenetic regulatory changes. The article above isn’t bad but they discount a third mode of reproduction in hyphal fungi – fragmentation. This is especially important in the lab where we propagate cultures by fragmentation but in nature too. Imagine how many hyphal fragments get moved around in a flash flood when fast running water washes over, around, and through mycelial colonies. And as anyone that’s had much experience at propagation of fungal colonies will tell you new ones start up much faster from a vegetative hyphal fragment than they do from spore germination.

    Your move, Bob. But personally, if I had a doctorate in mycology and was getting my ass kicked like you are in my own expertise by an unlettered google crazy autodidact I’d ditch my handle and start over somewhere else. But hey, that’s just me.

  16. All this talk about budding mushrooms is making me hungry. I’m heading out for a mushroom pizza and beer, if anyone wants to join me.

    Seriously though, nice work Dave. Keep us posted as your experimentation continues.

  17. DS,

    Re RM + NS, you say:

    ” I’m still looking for the first thing it can explain other than death of the individual and extinction of the species.”

    I say, learn about the Luria-Delbruck experiment:

    http://en.wikipedia.org/wiki/L.....experiment

    The Luria-Delbruck experiment proves that bacteria have fast acting defense mechanisms that operate far too quickly for constant rate random mutation to account for. If everything in bacterial genomes mutated at the rate Luria observed they’d all die of deleterious mutations to genes critical to normal metabolism and reproduction. Recent work by Scripps researchers have uncovered some bits of these defense mechanisms. I’m not sure what your point is in any case. LD only showed that changes can occur in the absence of selection pressure. One might also interpret the result as showing that bacteria anticipate the need for variation in defense mechanisms and constantly vary such defenses so that when an unknown threat shows up some of the colony will be pre-equipped to survive. This is called the “shotgun” method in engineering i.e. if you don’t know exactly how to solve something try throwing multiple solutions at the problem until you find something that works. -ds

  18. DS, can explain how the new understanding of epigenetics has anything to do with ID ?

    I’m not sure that it does have anything to do with ID other than being in agreement with IDers that traditional thinking about the mechanisms underlying organic evolution are wrong. -ds

  19. Mycelia are colonies of discrete individual organisms.

    What do you mean by an individual? The usage I’m use to, and the one in the Genetics paper you cite, equates an individual with a colony: i.e. all of the cells are produced from a single individual, are interconnected, and so you can get signals passed between the cells: in other words, in this indivudual, the induction was systemic. I don’t see any evidence from what you’ve written to suggest that more than one individual was involved, i.e. no evidence for inheritance. You would need to collect the spores and test them for peroxidase activity. Incidentally, if you try to investigate this further, you should also check the peroxide concentrations: you would have to rule out the explanation that the peroxide concentration had simply decreased by enough to allow normal growth.

    Tom Volk has a
    great website about fungi
    , including a page about the largest fungus known to man.

    Bob

    ]

    These are colonial organisms. Individual cells are separated by perforated junctions which cause them to be easily detachable from the colony without damage. Think of them like a roll of tickets with perforations between each ticket so they’re easily detachable. Each cell is independently capable of starting another colony and such fragmentation is a major mode of reproduction for these organisms. By individual in this case I mean a cell capable of living and reproducing independent of any other cell. In colonial organisms these cells cooperate for mutual benefit but each remains capable of independence. Indeed, some fungi are capable of both perforate hyphal growth and completely separate growth. In any case, the colony grows by mitosis. Anytime you have cellular division the chance arises for the DNA in the daughter cell to be different from the parent through a litany of different copy errors which I hope I don’t need to spell out for you. Thus descent with genetic modification occurs even during asexual vegetative growth and since each individual cell is capable of starting its own colony then traditional descent with modification is a reality. Indeed, if a modification occurs in a single cell it’s likely that it will continue mitotic cell division within the colony and all the subsequent hyphal branching from that cell will carry any mutation. Epigenetic changes propagate in a similar manner. Perhaps you should look at it this way. The colony that is now (ostensibly) tolerant of h202 is no longer the same as the colony from which it was cloned by fragmentation. Indeed, I cloned many new colonies via fragmentation and most of them died in lower peroxide environments. The fact that one survived, and it survived in the highest peroxide environment tested, is indicative of a fundamental change of some sort that occured in that colony. Of course further testing to eliminate other possibilities is required. I’m doing that as we speak. The first thing I did was reculture both the control non-peroxide-tolerant colony and the new peroxide tolerant colony onto fresh PDA plates without peroxide. This was to isolate to a single variable (hypothetical peroxide tolerance) and to insure that no overt genetic damage occured to the colony that survived the high peroxide environment. That step is accomplished and I can’t detect any difference. I now have 8 healthy colonies, 4 each of the control and peroxide tolerant, indistinguishable except for the labels. The next step is to reculture both the tolerant and non-tolerant cultures onto peroxide containing agar. If there was a persistant heritable change I should see a marked difference in growth rates. I wish I hadn’t been testing hydrogels at the same time I was testing for peroxide tolerance as that complicated things but it’s just SO easy to vary the composition of the substrate with hydrogel vs. agar I decided to try two variables at once. I really didn’t expect this result. As others have indicated it’s possible that peroxide decomposition is what happened but it’s unlikely as peroxide decomposition would have been gradual and the change in growth was instant – it went from absolutely no growth for two weeks to normal growth with no transition phase. There should have been an accelerating growth rate observed if decomposition was a factor. -ds

  20. Why was a mushroom the life of the party? He was a fungi.

  21. Bob, if you sent another comment since the above it wasn’t deleted purposefully. It may have contained a blacklisted spam word and got deleted along with the spam which has been incoming at such a high rate the past several days I haven’t been going through it all searching for mistakes. I normally look through the comments that have been tagged as spam looking for the 1% that isn’t spam but there’s usually only 100 of them each day to look through and this past week that suddenly jumped to 1000 per day. Still, I’ve been looking at all of them (I thought) but I just yesterday noticed a bug in the spam filter (Akismet plugin). If there are more than 150 comments awaiting human review only the first 150 are displayed. In the past week there have been many times when there were well over 200 comments in the filter and there is no way to look at the overflow so I’ve been flushing a lot of them without ever seeing them. My apologies if yours was one of them.

    On further consideration, I removed your name from the moderation list altogether. This won’t stop comments with blacklisted words from being intercepted but it does mean that those without blacklisted words will post immediately.

  22. Update: One of the four new colonies started from the peroxide tolerant colony has contaminated at the inoculation point with one of the two common fungal contaminants I get here. The two common ones are penicillium and another unidentified mold or even oyster mushroom with white cottony mycelia that quickly becomes saturated with black conidiospores. The new colonies are on antibiotic agar without peroxide to inhibit fungal contaminants. Since I did the reculturing carefully in a HEPA laminar flow hood there’s very little chance that the contaminant spores weren’t already present in the peroxide tolerant colony (which was purposely cultured in a non-sterile environment) but was being suppressed by the peroxide.

    Curious, I just checked the original culture which, after being hacked up to make the new cultures, I left sitting on top of the laminar flow hood in indirect sunlight. It’s been there one week and it’s a month old altogether (the volvariella took off like a gunshot after two weeks). It now has a tiny penicillium colony on it the size of a matchhead. The penicillium is mature, completely green, with hardly any active white mycelia at the edge of the colony. That’s not normal for pencillium with lots of room to grow on a nutrient rich substrate. It’s still being suppressed by peroxide which even after a month, including a week near a sunny window, is still present in the hydrogel. It’ll be interesting to see how long the pencillium takes to spread out. The small colony has gone to spore so the environment inside that culture container is just swimming with penicillium spores and a nutrient rich substrate. There is no new growth on what’s left of the volvariella in the original culture but that’s not unusual as it doesn’t grow well at 75F (normal incubation temp is 90F) and at two weeks old is losing its vigor too.

  23. (Curses, I’ve forgotten what I had written…)

    I see you haven’t preented any evidence that the colony is more than one individual. Even if an individual cell has the potential to form a new colony, that doesn’t mean it is a different indiviudual: there will still be coordination and signalling between cells of the colony, so they’re acting as one individual.

    You now no longer seem to believe that the change was due to a mutation (in the genetic sense), so our only dispute appears to be whether the colony is one individual or not (if it is one individual, then you clearly can’t have inheritance, unless you want to stretch the meanin of that word to breaking point too). Obviously we’re not going to agree on this.

    Bob

    It’s a colonial organism. You used the example of your right and left arms being parts of one individual. If it rains hard will bits of your arms flake off and grow into new Bobs? :roll: Are you saying that organisms can’t evolve through mitosis when the cells that are splitting are capable of independent reproduction? I need you to drive a stake in the ground on these questions, Bob. When you do, your argument is lost about whether evolution can take place in perforate mycelia through vegetative branching of the hyphae. I never claimed that anything more than a heritable regulatory change in enzyme expression from an epigenetic mechanism has taken place, even though genetic mutation IS possible, and I’m not even certain about that yet as still I need to 1) determine that the change is sticky (does it persist for long in the absence of the peroxide?) and 2) did the peroxide decompose and the colony never became tolerant. There are many reasons to think the peroxide didn’t decompose but there will be further testing to rule that out. I think it was epigenetic and transitory (but probably sticky for a good number of generations) because enzyme regulation in direct response to the environment where it persists for a number of generations after enzymes are no longer needed is common knowledge in mushroom tissue culture. Many mushrooms, especially wood decomposers, can digest a wide variety of substrates if given some time to adjust their enzyme production. When maintaining pure tissue cultures of commercial strains on agar it is common practice to put small amounts of their intended fruiting substrate into the agar so the culture maintains the enzyme compliment needed to digest it. I expect peroxide tolerance works the same way as peroxidases and the quantity produced is just one more extra-cellular enzyme these fungi employ to make a living. -ds

  24. Compare and contrast…
    From the main post:
    “So anyhow, I now have a volvariella colony that mutated beneficially …” (emphasis mine)

    From reply to my comment 23:
    “I never claimed that anything more than a heritable regulatory change in enzyme expression from an epigenetic mechanism has taken place,…”

    And I guess you’re going to continue to avoid the question about whether the fungus is one organism.

    Bob

    Mutation = change = modification. Mitotic cell division = descended cell. Descent with modification = evolution. The colony evolved. That’s my claim. I could be wrong. There are more experiments to conduct to confirm or falsify the hypothesis. One colonial organism. Many individual cells capable of independent life compose the colony. That’s not an individual in the normal sense of the word. It’s a colony.

    You also avoided my questions. That’s hardly fair as I’m answering yours. Please answer these:

    1) You used your right and left arms as examples of parts of the same invidual. If it rains hard will bits of your arms flake off and grow into new Bobs?

    2) Are you saying that organisms can’t evolve through mitosis when the cells that are splitting are capable of independent reproduction?

    Fair warning: If your next comment doesn’t answer these then it won’t show up. -ds

  25. Your first question deserves to be ignore with disdain: the answer is obvious, and beside the point I was trying to make: I was making a point about what the organism was actually doing, not what it might do. Your original post was about what the organism had actually done, nut what it might have done.

    To answer your second question, organisms can’t evolve (in a Darwinian sense). That sort of change within an organism is called development.

    Bob

    No Bob, it’s called evolution. This isn’t an egg turning into a mature organism. It’s a vegetative colony happily reproducing asexually. We’ll have to agree to disagree. You can go home now. Come back again soon, but not too soon. -ds

Leave a Reply